System for cell-based screening

ABSTRACT

The present invention provides systems, methods, screens, reagents and kits for optical system analysis of cells to rapidly determine the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for those that specifically affect particular biological functions.

CROSS REFERENCE

[0001] This application claims priority to U.S. Provisional Applicationsfor Patent Serial No. 60/122,152 (Feb. 26, 1999), No. 60/123,399 (Mar.8, 1999), No. 60/151,797 (Aug. 31, 1999), No. 60/168,408 (Dec. 1, 1999);and is a continuation in part of Ser. No. 09/430,656 (Oct. 29, 1999);Ser. No. 09/398,965 filed Sep. 17, 1999 which is a continuation in partof Ser. No. 09/352,171 filed Jul. 12, 1999, which is a continuation inpart of Ser. No. 09/031,271 filed Feb. 27, 1998 which is a continuationin part of U.S. application Ser. No. 08/810,983, filed on Feb. 27, 1997.

STATEMENT OF GOVERNMENT RIGHTS

[0002] This invention was made with Government support under ContractNo. N00014-98-C-0326 awarded by the Office of Naval Research, and thusthe Government may have certain rights in the invention.

FIELD OF THE INVENTION

[0003] This invention is in the field of fluorescence-based cell andmolecular biochemical assays for drug discovery.

BACKGROUND OF THE INVENTION

[0004] Drug discovery, as currently practiced in the art, is a long,multiple step process involving identification of specific diseasetargets, development of an assay based on a specific target, validationof the assay, optimization and automation of the assay to produce ascreen, high throughput screening of compound libraries using the assayto identify “hits”, hit validation and hit compound optimization. Theoutput of this process is a lead compound that goes into pre-clinicaland, if validated, eventually into clinical trials. In this process, thescreening phase is distinct from the assay development phases, andinvolves testing compound efficacy in living biological systems.

[0005] Historically, drug discovery is a slow and costly process,spanning numerous years and consuming hundreds of millions of dollarsper drug created. Developments in the areas of genomics and highthroughput screening have resulted in increased capacity and efficiencyin the areas of target identification and volume of compounds screened.Significant advances in automated DNA sequencing, PCR application,positional cloning, hybridization arrays, and bioinformatics havegreatly increased the number of genes (and gene fragments) encodingpotential drug screening targets. However, the basic scheme for drugscreening remians the same.

[0006] Validation of genomic targets as points for therapeuticintervention using the existing methods and protocols has become abottleneck in the drug discovery process due to the slow, manual methodsemployed, such as in vivo functional models, functional analysis ofrecombinant proteins, and stable cell line expression of candidategenes. Primary DNA sequence data acquired through automated sequencingdoes not permit identification of gene function, but can provideinformation about common “motifs” and specific gene homology whencompared to known sequence databases. Genomic methods such assubtraction hybridization and RADE (rapid amplification of differentialexpression) can be used to identify genes that are up or down regulatedin a disease state model. However, identification and validation stillproceed down the same pathway. Some proteomic methods use proteinidentification (global expression arrays, 2D electrophoresis,combinatorial libraries) in combination with reverse genetics toidentify candidate genes of interest. Such putative “disease associatedsequences” or DAS isolated as intact cDNA are a great advantage to thesemethods, but they are identified by the hundreds without providing anyinformation regarding type, activity, and distribution of the encodedprotein. Choosing a subset of DAS as drug screening targets is “random”,and thus extremely inefficient, without functional data to provide amechanistic link with disease. It is necessary, therefore, to providenew technologies to rapidly screen DAS to establish biological function,thereby improving target validation and candidate optimization in drugdiscovery.

[0007] There are three major avenues for improving early drug discoveryproductivity. First, there is a need for tools that provide increasedinformation handling capability. Bioinformatics has blossomed with therapid development of DNA sequencing systems and the evolution of thegenomics database. Genomics is beginning to play a critical role in theidentification of potential new targets. Proteomics has becomeindispensible in relating structure and function of protein targets inorder to predict drug interactions. However, the next level ofbiological complexity is the cell. Therefore, there is a need toacquire, manage and search multi-dimensional information from cells.Secondly, there is a need for higher throughput tools. Automation is akey to improving productivity as has already been demonstrated in DNAsequencing and high throughput primary screening. The instant inventionprovides for automated systems that extract multiple parameterinformation from cells that meet the need for higher throughput tools.The instant invention also provides for miniaturizing the methods,thereby allowing increased throughput, while decreasing the volumes ofreagents and test compounds required in each assay.

[0008] Radioactivity has been the dominant read-out in early drugdiscovery assays. However, the need for more information, higherthroughput and miniaturization has caused a shift towards usingfluorescence detection. Fluorescence-based reagents can yield morepowerful, multiple parameter assays that are higher in throughput andinformation content and require lower volumes of reagents and testcompounds. Fluorescence is also safer and less expensive thanradioactivity-based methods.

[0009] Screening of cells treated with dyes and fluorescent reagents iswell known in the art. There is a considerable body of literaturerelated to genetic engineering of cells to produce fluorescent proteins,such as modified green fluorescent protein (GFP), as a reportermolecule. Some properties of wild-type GFP are disclosed by Morise etal. (Biochemistry 13 (1974), p. 2656-2662), and Ward et al. (Photochem.Photobiol. 31 (1980), p. 611-615). The GFP of the jellyfish AequoreaVictoria has an excitation maximum at 395 nm and an emission maximum at510 nm, and does not require an exogenous factor for fluorescenceactivity. Uses for GFP disclosed in the literature are widespread andinclude the study of gene expression and protein localization (Chalfieet al., Science 263 (1994), p. 12501-12504)), as a tool for visualizingsubcellular organelles (Rizzuto et al., Curr. Biology 5 (1995), p.635-642)), visualization of protein transport along the secretorypathway (Kaether and Gerdes, FEBS Letters 369 (1995), p. 267-271)),expression in plant cells (Hu and Cheng, FEBS Letters 369 (1995), p.331-334)) and Drosophila embryos (Davis et al., Dev. Biology 170 (1995),p. 726-729)), and as a reporter molecule fused to another protein ofinterest (U.S. Pat. No. 5,491,084). Similarly, WO96/23898 relates tomethods of detecting biologically active substances affectingintracellular processes by utilizing a GFP construct having a proteinkinase activation site. This patent, and all other patents referenced inthis application are incorporated by reference in their entirety

[0010] Numerous references are related to GFP proteins in biologicalsystems. For example, WO 96/09598 describes a system for isolating cellsof interest utilizing the expression of a GFP like protein. WO 96/27675describes the expression of GFP in plants. WO 95/21191 describesmodified GFP protein expressed in transformed organisms to detectmutagenesis. U.S. Pat. Nos. 5,401,629 and 5,436,128 describe assays andcompositions for detecting and evaluating the intracellular transductionof an extracellular signal using recombinant cells that express cellsurface receptors and contain reporter gene constructs that includetranscriptional regulatory elements that are responsive to the activityof cell surface receptors.

[0011] Performing a screen on many thousands of compounds requiresparallel handling and processing of many compounds and assay componentreagents. Standard high throughput screens (“HTS”) use mixtures ofcompounds and biological reagents along with some indicator compoundloaded into arrays of wells in standard microtiter plates with 96 or 384wells. The signal measured from each well, either fluorescence emission,optical density, or radioactivity, integrates the signal from all thematerial in the well giving an overall population average of all themolecules in the well.

[0012] Science Applications International Corporation (SAIC) 130 FifthAvenue, Seattle, Wash. 98109) describes an imaging plate reader. Thissystem uses a CCD camera to image the whole area of a 96 well plate. Theimage is analyzed to calculate the total fluorescence per well for allthe material in the well.

[0013] Molecular Devices, Inc. (Sunnyvale, Calif.) describes a system(FLIPR) which uses low angle laser scanning illumination and a mask toselectively excite fluorescence within approximately 200 microns of thebottoms of the wells in standard 96 well plates in order to reducebackground when imaging cell monolayers. This system uses a CCD camerato image the whole area of the plate bottom. Although this systemmeasures signals originating from a cell monolayer at the bottom of thewell, the signal measured is averaged over the area of the well and istherefore still considered a measurement of the average response of apopulation of cells. The image is analyzed to calculate the totalfluorescence per well for cell-based assays. Fluid delivery devices havealso been incorporated into cell based screening systems, such as theFLIPR system, in order to initiate a response, which is then observed asa whole well population average response using a macro-imaging system.

[0014] In contrast to high throughput screens, various high-contentscreens (“HCS”) have been developed to address the need for moredetailed information about the temporal-spatial dynamics of cellconstituents and processes. High-content screens automate the extractionof multicolor fluorescence information derived from specificfluorescence-based reagents incorporated into cells (Giuliano and Taylor(1995), Curr. Op. Cell Biol. 7:4; Giuliano et al. (1995) Ann. Rev.Biophys. Biomol. Struct. 24:405). Cells are analyzed using an opticalsystem that can measure spatial, as well as temporal dynamics. (Farkaset al. (1993) Ann. Rev. Physiol. 55:785; Giuliano et al. (1990) InOptical Microscopy for Biology. B. Herman and K. Jacobson (eds.), pp.543-557. Wiley-Liss, New York; Hahn et al (1992) Nature 359:736;Waggoner et al. (1996) Hum. Pathol. 27:494). The concept is to treateach cell as a “well” that has spatial and temporal information on theactivities of the labeled constituents.

[0015] The types of biochemical and molecular information now accessiblethrough fluorescence-based reagents applied to cells include ionconcentrations, membrane potential, specific translocations, enzymeactivities, gene expression, as well as the presence, amounts andpatterns of metabolites, proteins, lipids, carbohydrates, and nucleicacid sequences (DeBiasio et al., (1996) Mol. Biol. Cell. 7:1259;Giulianoet al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405; Heim andTsien, (1996) Curr. Biol. 6:178).

[0016] High-content screens can be performed on either fixed cells,using fluorescently labeled antibodies, biological ligands, and/ornucleic acid hybridization probes, or live cells using multicolorfluorescent indicators and “biosensors.” The choice of fixed or livecell screens depends on the specific cell-based assay required.

[0017] Fixed cell assays are the simplest, since an array of initiallyliving cells in a microtiter plate format can be treated with variouscompounds and doses being tested, then the cells can be fixed, labeledwith specific reagents, and measured. No environmental control of thecells is required after fixation. Spatial information is acquired, butonly at one time point. The availability of thousands of antibodies,ligands and nucleic acid hybridization probes that can be applied tocells makes this an attractive approach for many types of cell-basedscreens. The fixation and labeling steps can be automated, allowingefficient processing of assays.

[0018] Live cell assays are more sophisticated and powerful, since anarray of living cells containing the desired reagents can be screenedover time, as well as space. Environmental control of the cells(temperature, humidity, and carbon dioxide) is required duringmeasurement, since the physiological health of the cells must bemaintained for multiple fluorescence measurements over time. There is agrowing list of fluorescent physiological indicators and “biosensors”that can report changes in biochemical and molecular activities withincells (Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct.24:405; Hahn et al., (1993) In Fluorescent and Luminescent Probes forBiological Activity. W. T. Mason, (ed.), pp. 349-359, Academic Press,San Diego).

[0019] The availability and use of fluorescence-based reagents hashelped to advance the development of both fixed and live cellhigh-content screens. Advances in instrumentation to automaticallyextract multicolor, high-content information has recently made itpossible to develop HCS into an automated tool. An article by Taylor, etal. (American Scientist 80 (1992), p. 322-335) describes many of thesemethods and their applications. For example, Proffitt et. al. (Cytometry24: 204-213 (1996)) describe a semi-automated fluorescence digitalimaging system for quantifying relative cell numbers in situ in avariety of tissue culture plate formats, especially 96-well microtiterplates. The system consists of an epifluorescence inverted microscopewith a motorized stage, video camera, image intensifier, and amicrocomputer with a PC-Vision digitizer. Turbo Pascal software controlsthe stage and scans the plate taking multiple images per well. Thesoftware calculates total fluorescence per well, provides for dailycalibration, and configures easily for a variety of tissue culture plateformats. Thresholding of digital images and reagents which fluoresceonly when taken up by living cells are used to reduce backgroundfluorescence without removing excess fluorescent reagent.

[0020] Scanning confocal microscope imaging (Go et al., (1997)Analytical Biochemistry 247:210-215; Goldman et al., (1995) ExperimentalCell Research 221:311-319) and multiphoton microscope imaging (Denk etal., (1990) Science 248:73; Gratton et al., (1994) Proc. of theMicroscopical Society of America, pp. 154-155) are also well establishedmethods for acquiring high resolution images of microscopic samples. Theprinciple advantage of these optical systems is the very shallow depthof focus, which allows features of limited axial extent to be resolvedagainst the background. For example, it is possible to resolve internalcytoplasmic features of adherent cells from the features on the cellsurface. Because scanning multiphoton imaging requires very shortduration pulsed laser systems to achieve the high photon flux required,fluorescence lifetimes can also be measured in these systems (Lakowiczet al., (1992) Anal. Biochem. 202:316-330; Gerrittsen et al. (1997), J.of Fluorescence 7:11-15)), providing additional capability for differentdetection modes. Small, reliable and relatively inexpensive lasersystems, such as laser diode pumped lasers, are now available to allowmultiphoton confocal microscopy to be applied in a fairly routinefashion.

[0021] A combination of the biological heterogeneity of cells inpopulations (Bright, et al., (1989). J. Cell. Physiol. 141:410;Giuliano, (1996) Cell Motil. Cytoskel. 35:237)) as well as the highspatial and temporal frequency of chemical and molecular informationpresent within cells, makes it impossible to extract high-contentinformation from populations of cells using existing whole microtiterplate readers. No existing high-content screening platform has beendesigned for multicolor, fluorescence-based screens using cells that areanalyzed individually. Similarly, no method is currently available thatcombines automated fluid delivery to arrays of cells for the purpose ofsystematically screening compounds for the ability to induce a cellularresponse that is identified by HCS analysis, especially from cells grownin microtiter plates. Furthermore, no method exists in the art combininghigh throughput well-by-well measurements to identify “hits” in oneassay followed by a second high content cell-by-cell measurement on thesame plate of only those wells identified as hits.

[0022] The instant invention provides systems, methods, and screens thatcombine high throughput screening (HTS) and high content screening (HCS)that significantly improve target validation and candidate optimizationby combining many cell screening formats with fluorescence-basedmolecular reagents and computer-based feature extraction, data analysis,and automation, resulting in increased quantity and speed of datacollection, shortened cycle times, and, ultimately, faster evaluation ofpromising drug candidates. The instant invention also provides forminiaturizing the methods, thereby allowing increased throughput, whiledecreasing the volumes of reagents and test compounds required in eachassay.

SUMMARY OF THE INVENTION

[0023] In one aspect, the present invention relates to a method foranalyzing cells comprising providing cells containing fluorescentreporter molecules in an array of locations, treating the cells in thearray of locations with one or more reagents, imaging numerous cells ineach location with fluorescence optics, converting the opticalinformation into digital data, utilizing the digital data to determinethe distribution, environment or activity of the fluorescently labeledreporter molecules in the cells and the distribution of the cells, andinterpreting that information in terms of a positive, negative or nulleffect of the compound being tested on the biological function

[0024] In this embodiment, the method rapidly determines thedistribution, environment, or activity of fluorescently labeled reportermolecules in cells for the purpose of screening large numbers ofcompounds for those that specifically affect particular biologicalfunctions. The array of locations may be a microtiter plate or amicrochip which is a microplate having cells in an array of locations.In a preferred embodiment, the method includes computerized means foracquiring, processing, displaying and storing the data received. In apreferred embodiment, the method further comprises automated fluiddelivery to the arrays of cells. In another preferred embodiment, theinformation obtained from high throughput measurements on the same plateare used to selectively perform high content screening on only a subsetof the cell locations on the plate.

[0025] In another aspect of the present invention, a cell screeningsystem is provided that comprises:

[0026] a high magnification fluorescence optical system having amicroscope objective,

[0027] an XY stage adapted for holding a plate containing an array ofcells and having a means for moving the plate for proper alignment andfocusing on the cell arrays;

[0028] a digital camera;

[0029] a light source having optical means for directing excitationlight to cell arrays and a means for directing fluorescent light emittedfrom the cells to the digital camera; and

[0030] a computer means for receiving and processing digital data fromthe digital camera wherein the computer means includes a digital framegrabber for receiving the images from the camera, a display for userinteraction and display of assay results, digital storage media for datastorage and archiving, and a means for control, acquisition, processingand display of results.

[0031] In a preferred embodiment, the cell screening system furthercomprises a computer screen operatively associated with the computer fordisplaying data. In another preferred embodiment, the computer means forreceiving and processing digital data from the digital camera stores thedata in a bioinformatics data base. In a further preferred embodiment,the cell screening system further comprises a reader that measures asignal from many or all the wells in parallel. In another preferredembodiment, the cell screening system further comprises amechanical-optical means for changing the magnification of the system,to allow changing modes between high throughput and high contentscreening. In another preferred embodiment, the cell screening systemfurther comprises a chamber and control system to maintain thetemperature, CO₂ concentration and humidity surrounding the plate atlevels required to keep cells alive. In a further preferred embodiment,the cell screening system utilizes a confocal scanning illumination anddetection system.

[0032] In another aspect of the present invention, a machine readablestorage medium comprising a program containing a set of instructions forcausing a cell screening system to execute procedures for defining thedistribution and activity of specific cellular constituents andprocesses is provided. In a preferred embodiment, the cell screeningsystem comprises a high magnification fluorescence optical system with astage adapted for holding cells and a means for moving the stage, adigital camera, a light source for receiving and processing the digitaldata from the digital camera, and a computer means for receiving andprocessing the digital data from the digital camera. Preferredembodiments of the machine readable storage medium comprise programsconsisting of a set of instructions for causing a cell screening systemto execute the procedures set forth in FIGS. 9, 11, 12, 13, 14 or 15.Another preferred embodiment comprises a program consisting of a set ofinstructions for causing a cell screening system to execute proceduresfor detecting the distribution and activity of specific cellularconstituents and processes. In most preferred embodiments, the cellularprocesses include, but are not limited to, nuclear translocation of aprotein, cellular hypertrophy, apoptosis, and protease-inducedtranslocation of a protein.

[0033] In another preferred embodiment, a variety of automated cellscreening methods are provided, including screens to identify compoundsthat affect transcription factor activity, protein kinase activity, cellmorphology, microtubule structure, apoptosis, receptor internalization,and protease-induced translocation of a protein.

[0034] In another aspect, the present invention provides recombinantnucleic acids encoding a protease biosensor, comprising:

[0035] a. a first nucleic acid sequence that encodes at least onedetectable polypeptide signal;

[0036] b. a second nucleic acid sequence that encodes at least oneprotease recognition site, wherein the second nucleic acid sequence isoperatively linked to the first nucleic acid sequence that encodes theat least one detectable polypeptide signal; and

[0037] c. a third nucleic acid sequence that encodes at least onereactant target sequence, wherein the third nucleic acid sequence isoperatively linked to the second nucleic acid sequence that encodes theat least one protease recognition site.

[0038] The present invention also provides the recombinant expressionvectors capable of expressing the recombinant nucleic acids encodingprotease biosensors, as well as genetically modified host cells that aretransfected with the expression vectors.

[0039] The invention further provides recombinant protease biosensors,comprising

[0040] a. a first domain comprising at least one detectable polypeptidesignal;

[0041] b. a second domain comprising at least one protease recognitionsite; and

[0042] c. a third domain comprising at least one reactant targetsequence;

[0043] wherein the first domain and the third domain are separated bythe second domain.

[0044] In a further aspect, the present invention involves assays andreagents for characterizing a sample for the presence of a toxin. Themethod comprises the use of detector, classifier, and identifier classesof toxin biosensors to provide for various levels of toxincharacterization.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045]FIG. 1 shows a diagram of the components of the cell-basedscanning system.

[0046]FIG. 2 shows a schematic of the microscope subassembly.

[0047]FIG. 3 shows the camera subassembly.

[0048]FIG. 4 illustrates cell scanning system process.

[0049]FIG. 5 illustrates a user interface showing major functions toguide the user.

[0050]FIG. 6 is a block diagram of the two platform architecture of theDual Mode System for Cell Based Screening in which one platform uses atelescope lens to read all wells of a microtiter plate and a secondplatform that uses a higher magnification lens to read individual cellsin a well.

[0051]FIG. 7 is a detail of an optical system for a single platformarchitecture of the Dual Mode System for Cell Based Screening that usesa moveable ‘telescope’ lens to read all wells of a microtiter plate anda moveable higher magnification lens to read individual cells in a well.

[0052]FIG. 8 is an illustration of the fluid delivery system foracquiring kinetic data on the Cell Based Screening System.

[0053]FIG. 9 is a flow chart of processing step for the cell-basedscanning system.

[0054] FIGS. 10A-J illustrates the strategy of the Nuclear TranslocationAssay.

[0055]FIG. 11 is a flow chart defining the processing steps in the DualMode System for Cell Based Screening combining high throughput and highcontent screening of microtiter plates.

[0056]FIG. 12 is a flow chart defining the processing steps in the HighThroughput mode of the System for Cell Based Screening.

[0057]FIG. 13 is a flow chart defining the processing steps in the HighContent mode of the System for Cell Based Screening.

[0058]FIG. 14 is a flow chart defining the processing steps required foracquiring kinetic data in the High Content mode of the System for CellBased Screening.

[0059]FIG. 15 is a flow chart defining the processing steps performedwithin a well during the acquisition of kinetic data.

[0060]FIG. 16 is an example of data from a known inhibitor oftranslocation.

[0061]FIG. 17 is an example of data from a known stimulator oftranslocation.

[0062]FIG. 18 illustrates data presentation on a graphical display.

[0063]FIG. 19 is an illustration of the data from the High Throughputmode of the System for Cell Based Screening, an example of the datapassed to the High Content mode, the data acquired in the high contentmode, and the results of the analysis of that data.

[0064]FIG. 20 shows the measurement of a drug-induced cytoplasm tonuclear translocation.

[0065]FIG. 21 illustrates a graphical user interface of the measurementshown in FIG. 20.

[0066]FIG. 22 illustrates a graphical user interface, with datapresentation, of the measurement shown in FIG. 20.

[0067]FIG. 23 is a graph representing the kinetic data obtained from themeasurements depicted in FIG. 20.

[0068]FIG. 24 details a high-content screen of drug-induced apoptosis.

[0069]FIG. 25. Graphs depicting changes in morphology upon induction ofapoptosis. Staurosporine (A) and paclitaxel (B) induce classic nuclearfragmentation in L929 cells. BHK cells exhibit concentration dependentchanges in response to staurosporine (C), but a more classical responseto paclitaxel (D). MCF-7 cells exhibit either nuclear condensation (E)or fragmentation (F) in response to staurosporine and paclitaxel,respectively. In all cases, cells were exposed to the compounds for 30hours.

[0070]FIG. 26 illustrates the dose response of cells to staurosporine interms of both nuclear size and nuclear perimeter convolution.

[0071]FIG. 27. Graphs depicting induction of apoptosis by staurosporineand paclitaxel leading to changes in peri-nuclear f-actin content. (A,B) Both apoptotic stimulators induce dose-dependent increases in f-actincontent in L929 cells. (C) In BHK cells, staurosporine induces adose-dependent increase in f-actin, whereas paclitaxel (D) producesresults that are more variable. (E) MCF-7 cells exhibit either adecrease or increase depending on the concentration of staurosporine.(F) Paclitaxel induced changes in f-actin content were highly variableand not significant. Cells were exposed to the compounds for 30 hours.

[0072]FIG. 28. Graphs depicting mitochondrial changes in response toinduction of apoptosis. L929 (A,B) and BHK (C,D) cells responded to bothstaurosporine (A,C) and paclitaxel (B,D) with increases in mitochondrialmass. MCF-7 cells exhibit either a decrease in membrane potential (E,staurosporine) or an increase in mitochondrial mass (F, paclitaxel)depending on the stimulus. Cells were exposed to the compounds for 30hours. 28G is a graph showing the simultaneous measurement ofstaurosporine effects on mitochondrial mass and mitochondrial potentialin BHK cells.

[0073]FIG. 29 shows the nucleic acid and amino acid sequence for varioustypes of protesae biosensor domains. (A) Signal sequences. (B) Proteaserecognition sites. (C) Product/Reactant target sequences

[0074]FIG. 30 shows schematically shows some basic organization ofdomains in the protease biosensors of the invention.

[0075]FIG. 31 is a schematic diagram of a specific 3-domain proteasebiosensor.

[0076]FIG. 32 is a photograph showing the effect of stimulation ofapoptosis by cis-platin on BHK cells transfected with an expressionvector that expresses the caspase biosensor shown in FIG. 32.

[0077]FIG. 33 is a schematic diagram of a specific 4-domain proteasebiosensor.

[0078]FIG. 34 is a schematic diagram of a specific 4-domain proteasebiosensor, containing a nucleolar localization signal.

[0079]FIG. 35 is a schematic diagram of a specific 5-domain proteasebiosensor.

[0080]FIG. 36 shows the differential response in a dual labeling assayof the p38 MAPK and NF-κB pathways across three model toxins and twodifferent cell types. Treatments marked with an asterisk are differentfrom controls at a 99% confidence level (p<0.01).

DETAILED DESCRIPTION OF THE INVENTION

[0081] All cited patents, patent applications and other references arehereby incorporated by reference in their entirety.

[0082] As used herein, the following terms have the specified meaning:

[0083] Markers of cellular domains. Luminescent probes that have highaffinity for specific cellular constituents including specificorganelles or molecules. These probes can either be small luminescentmolecules or fluorescently tagged macromolecules used as “labelingreagents”, “environmental indicators”, or “biosensors.”

[0084] Labeling reagents. Labeling reagents include, but are not limitedto, luminescently labeled macromolecules including fluorescent proteinanalogs and biosensors, luminescent macromolecular chimeras includingthose formed with the green fluorescent protein and mutants thereof,luminescently labeled primary or secondary antibodies that react withcellular antigens involved in a physiological response, luminescentstains, dyes, and other small molecules.

[0085] Markers of cellular translocations. Luminescently taggedmacromolecules or organelles that move from one cell domain to anotherduring some cellular process or physiological response. Translocationmarkers can either simply report location relative to the markers ofcellular domains or they can also be “biosensors” that report somebiochemical or molecular activity as well.

[0086] Biosensors. Macromolecules consisting of a biological functionaldomain and a luminescent probe or probes that report the environmentalchanges that occur either internally or on their surface. A class ofluminescently labeled macromolecules designed to sense and report thesechanges have been termed “fluorescent-protein biosensors”. The proteincomponent of the biosensor provides a highly evolved molecularrecognition moiety. A fluorescent molecule attached to the proteincomponent in the proximity of an active site transduces environmentalchanges into fluorescence signals that are detected using a system withan appropriate temporal and spatial resolution such as the cell scanningsystem of the present invention. Because the modulation of nativeprotein activity within the living cell is reversible, and becausefluorescent-protein biosensors can be designed to sense reversiblechanges in protein activity, these biosensors are essentially reusable.

[0087] Disease associated sequences (“DAS”). This term refers to nucleicacid sequences identified by standard techniques, such as primary DNAsequence data, genomic methods such as subtraction hybridization andRADE, and proteomic methods in combination with reverse genetics, asbeing of drug candidate compounds. The term does not mean that thesequence is only associated with a disease state.

[0088] High content screening (HCS) can be used to measure the effectsof drugs on complex molecular events such as signal transductionpathways, as well as cell functions including, but not limited to,apoptosis, cell division, cell adhesion, locomotion, exocytosis, andcell-cell communication. Multicolor fluorescence permits multipletargets and cell processes to be assayed in a single screen.Cross-correlation of cellular responses will yield a wealth ofinformation required for target validation and lead optimization.

[0089] In one aspect of the present invention, a cell screening systemis provided comprising a high magnification fluorescence optical systemhaving a microscope objective, an XY stage adapted for holding a platewith an array of locations for holding cells and having a means formoving the plate to align the locations with the microscope objectiveand a means for moving the plate in the direction to effect focusing; adigital camera; a light source having optical means for directingexcitation light to cells in the array of locations and a means fordirecting fluorescent light emitted from the cells to the digitalcamera; and a computer means for receiving and processing digital datafrom the digital camera wherein the computer means includes: a digitalframe grabber for receiving the images from the camera, a display foruser interaction and display of assay results, digital storage media fordata storage and archiving, and means for control, acquisition,processing and display of results.

[0090]FIG. 1 is a schematic diagram of a preferred embodiment of thecell scanning system. An inverted fluorescence microscope is used 1,such as a Zeiss Axiovert inverted fluorescence microscope which usesstandard objectives with magnification of 1-100× to the camera, and awhite light source (e.g. 100W mercury-arc lamp or 75W xenon lamp) withpower supply 2. There is an XY stage 3 to move the plate 4 in the XYdirection over the microscope objective. A Z-axis focus drive 5 movesthe objective in the Z direction for focusing. A joystick 6 provides formanual movement of the stage in the XYZ direction. A high resolutiondigital camera 7 acquires images from each well or location on theplate. There is a camera power supply 8, an automation controller 9 anda central processing unit 10. The PC 11 provides a display 12 and hasassociated software. The printer 13 provides for printing of a hard copyrecord.

[0091]FIG. 2 is a schematic of one embodiment of the microscope assembly1 of the invention, showing in more detail the XY stage 3, Z-axis focusdrive 5, joystick 6, light source 2 and automation controller 9. Cablesto the computer 15 and microscope 16, respectively, are provided. Inaddition, FIG. 2 shows a 96 well microtiter plate 17 which is moved onthe XY stage 3 in the XY direction. Light from the light source 2 passesthrough the PC controlled shutter 18 to a motorized filter wheel 19 withexcitation filters 20. The light passes into filter cube 25 which has adichroic mirror 26 and an emission filter 27. Excitation light reflectsoff the dichroic mirror to the wells in the microtiter plate 17 andfluorescent light 28 passes through the dichroic mirror 26 and theemission filter 27 and to the digital camera 7.

[0092]FIG. 3 shows a schematic drawing of a preferred camera assembly.The digital camera 7, which contains an automatic shutter for exposurecontrol and a power supply 31 receives fluorescent light 28 from themicroscope assembly. A digital cable 30 transports digital signals tothe computer.

[0093] The standard optical configurations described above usemicroscope optics to directly produce an enlarged image of the specimenon the camera sensor in order to capture a high resolution image of thespecimen. This optical system is commonly referred to as ‘wide field’microscopy. Those skilled in the art of microscopy will recognize that ahigh resolution image of the specimen can be created by a variety ofother optical systems, including, but not limited to, standard scanningconfocal detection of a focused point or line of illumination scannedover the specimen (Go et al. 1997, supra), and multi-photon scanningconfocal microscopy (Denk et al., 1990, supra), both of which can formimages on a CCD detector or by synchronous digitization of the analogoutput of a photomultiplier tube.

[0094] In screening applications, it is often necessary to use aparticular cell line, or primary cell culture, to take advantage ofparticular features of those cells. Those skilled in the art of cellculture will recognize that some cell lines are contact inhibited,meaning that they will stop growing when they become surrounded by othercells, while other cell lines will continue to grow under thoseconditions and the cells will literally pile up, forming many layers. Anexample of such a cell line is the HEK 293 (ATCC CRL-1573) line. Anoptical system that can acquire images of single cell layers inmultilayer preparations is required for use with cell lines that tend toform layers. The large depth of field of wide field microscopes producesan image that is a projection through the many layers of cells, makinganalysis of subcellular spatial distributions extremely difficult inlayer-forming cells. Alternatively, the very shallow depth of field thatcan be achieved on a confocal microscope, (about one micron), allowsdiscrimination of a single cell layer at high resolution, simplifyingthe determination of the subcellular spatial distribution. Similarly,confocal imaging is preferable when detection modes such as fluorescencelifetime imaging are required.

[0095] The output of a standard confocal imaging attachment for amicroscope is a digital image that can be converted to the same formatas the images produced by the other cell screening system embodimentsdescribed above, and can therefore be processed in exactly the same wayas those images. The overall control, acquisition and analysis in thisembodiment is essentially the same. The optical configuration of theconfocal microscope system, is essentially the same as that describedabove, except for the illuminator and detectors. Illumination anddetection systems required for confocal microscopy have been designed asaccessories to be attached to standard microscope optical systems suchas that of the present invention (Zeiss, Germany). These alternativeoptical systems therefore can be easily integrated into the system asdescribed above.

[0096]FIG. 4 illustrates an alternative embodiment of the invention inwhich cell arrays are in microwells 40 on a microplate 41, described ionco-pending U.S. Application Ser. No. 08/865,341, incorporated byreference herein in its entirety. Typically the microplate is 20 mm by30 mm as compared to a standard 96 well microtiter plate which is 86 mmby 129 mm. The higher density array of cells on a microplate allows themicroplate to be imaged at a low resolution of a few microns per pixelfor high throughput and particular locations on the microplate to beimaged at a higher resolution of less than 0.5 microns per pixel. Thesetwo resolution modes help to improve the overall throughput of thesystem.

[0097] The microplate chamber 42 serves as a microfluidic deliverysystem for the addition of compounds to cells. The microplate 41 in themicroplate chamber 42 is placed in an XY microplate reader 43. Digitaldata is processed as described above. The small size of this microplatesystem increases throughput, minimizes reagent volume and allows controlof the distribution and placement of cells for fast and precisecell-based analysis. Processed data can be displayed on a PC screen 11and made part of a bioinformatics data base 44. This data base not onlypermits storage and retrieval of data obtained through the methods ofthis invention, but also permits acquisition and storage of externaldata relating to cells. FIG. 5 is a PC display which illustrates theoperation of the software.

[0098] In an alternative embodiment, a high throughput system (HTS) isdirectly coupled with the HCS either on the same platform or on twoseparate platforms connected electronically (e.g. via a local areanetwork). This embodiment of the invention, referred to as a dual modeoptical system, has the advantage of increasing the throughput of a HCSby coupling it with a HTS and thereby requiring slower high resolutiondata acquisition and analysis only on the small subset of wells thatshow a response in the coupled HTS.

[0099] High throughput ‘whole plate’ reader systems are well known inthe art and are commonly used as a component of an HTS system used toscreen large numbers of compounds (Beggs (1997), J. of Biomolec.Screening 2:71-78; Macaffrey et al., (1996) J. Biomolec. Screening1:187-190).

[0100] In one embodiment of dual mode cell based screening, a twoplatform architecture in which high throughput acquisition occurs on oneplatform and high content acquisition occurs on a second platform isprovided (FIG. 6). Processing occurs on each platform independently,with results passed over a network interface, or a single controller isused to process the data from both platforms.

[0101] As illustrated in FIG. 6, an exemplified two platform dual modeoptical system consists of two light optical instruments, a highthroughput platform 60 and a high content platform 65 which readfluorescent signals emitted from cells cultured in microtiter plates ormicrowell arrays on a microplate, and communicate with each other via anelectronic connection 64. The high throughput platform 60 analyzes allthe wells in the whole plate either in parallel or rapid serial fashion.Those skilled in the art of screening will recognize that there are amany such commercially available high throughput reader systems thatcould be integrated into a dual mode cell based screening system(Topcount (Packard Instruments, Meriden, Conn.); Spectramax, Lumiskan(Molecular Devices, Sunnyvale, Calif.); Fluoroscan (Labsystems, Beverly,Mass.)). The high content platform 65, as described above, scans fromwell to well and acquires and analyzes high resolution image datacollected from individual cells within a well.

[0102] The HTS software, residing on the system's computer 62, controlsthe high throughput instrument, and results are displayed on the monitor61. The HCS software, residing on it's computer system 67 controls thehigh content instrument hardware 65, optional devices (e.g. plateloader, environmental chamber, fluid dispenser), analyzes digital imagedata from the plate, displays results on the monitor 66 and manages datameasured in an integrated database. The two systems can also share asingle computer, in which case all data would be collected, processedand displayed on that computer, without the need for a local areanetwork to transfer the data. Microtiter plates are transferred from thehigh throughput system to the high content system 63 either manually orby a robotic plate transfer device, as is well known in the art (Beggs(1997), supra; Mcaffrey (1996), supra).

[0103] In a preferred embodiment, the dual mode optical system utilizesa single platform system (FIG. 7). It consists of two separate opticalmodules, an HCS module 203 and an HTS module 209 that can beindependently or collectively moved so that only one at a time is usedto collect data from the microtiter plate 201. The microtiter plate 201is mounted in a motorized X,Y stage so it can be positioned for imagingin either HTS or HCS mode. After collecting and analyzing the HTS imagedata as described below, the HTS optical module 209 is moved out of theoptical path and the HCS optical module 203 is moved into place.

[0104] The optical module for HTS 209 consists of a projection lens 214,excitation wavelength filter 213 and dichroic mirror 210 which are usedto illuminate the whole bottom of the plate with a specific wavelengthband from a conventional microscope lamp system (not illustrated). Thefluorescence emission is collected through the dichroic mirror 210 andemission wavelength filter 211 by a lens 212 which forms an image on thecamera 216 with sensor 215.

[0105] The optical module for HCS 203 consists of a projection lens 208,excitation wavelength filter 207 and dichroic mirror 204 which are usedto illuminate the back aperture of the microscope objective 202, andthereby the field of that objective, from a standard microscopeillumination system (not shown). The fluorescence emission is collectedby the microscope objective 202, passes through the dichroic mirror 204and emission wavelength filter 205 and is focused by a tube lens 206which forms an image on the same camera 216 with sensor 215.

[0106] In an alternative embodiment of the present invention, the cellscreening system further comprises a fluid delivery device for use withthe live cell embodiment of the method of cell screening (see below).FIG. 8 exemplifies a fluid delivery device for use with the system ofthe invention. It consists of a bank of 12 syringe pumps 701 driven by asingle motor drive. Each syringe 702 is sized according to the volume tobe delivered to each well, typically between 1 and 100 μL. Each syringeis attached via flexible tubing 703 to a similar bank of connectorswhich accept standard pipette tips 705. The bank of pipette tips areattached to a drive system so they can be lowered and raised relative tothe microtiter plate 706 to deliver fluid to each well. The plate ismounted on an X,Y stage, allowing movement relative to the opticalsystem 707 for data collection purposes. This set-up allows one set ofpipette tips, or even a single pipette tip, to deliver reagent to allthe wells on the plate. The bank of syringe pumps can be used to deliverfluid to 12 wells simultaneously, or to fewer wells by removing some ofthe tips.

[0107] In another aspect, the present invention provides a method foranalyzing cells comprising providing an array of locations which containmultiple cells wherein the cells contain one or more fluorescentreporter molecules; scanning multiple cells in each of the locationscontaining cells to obtain fluorescent signals from the fluorescentreporter molecule in the cells; converting the fluorescent signals intodigital data; and utilizing the digital data to determine thedistribution, environment or activity of the fluorescent reportermolecule within the cells.

[0108] Cell Arrays

[0109] Screening large numbers of compounds for activity with respect toa particular biological function requires preparing arrays of cells forparallel handling of cells and reagents. Standard 96 well microtiterplates which are 86 mm by 129 mm, with 6 mm diameter wells on a 9 mmpitch, are used for compatibility with current automated loading androbotic handling systems. The microplate is typically 20 mm by 30 mm,with cell locations that are 100-200 microns in dimension on a pitch ofabout 500 microns. Methods for making microplates are described in U.S.patent application Ser. No. 08/865,341, incorporated by reference hereinin its entirety. Microplates may consist of coplanar layers of materialsto which cells adhere, patterned with materials to which cells will notadhere, or etched 3-dimensional surfaces of similarly patteredmaterials. For the purpose of the following discussion, the terms ‘well’and ‘microwell’ refer to a location in an array of any construction towhich cells adhere and within which the cells are imaged. Microplatesmay also include fluid delivery channels in the spaces between thewells. The smaller format of a microplate increases the overallefficiency of the system by minimizing the quantities of the reagents,storage and handling during preparation and the overall movementrequired for the scanning operation. In addition, the whole area of themicroplate can be imaged more efficiently, allowing a second mode ofoperation for the microplate reader as described later in this document.

[0110] Fluorescence Reporter Molecules

[0111] A major component of the new drug discovery paradigm is acontinually growing family of fluorescent and luminescent reagents thatare used to measure the temporal and spatial distribution, content, andactivity of intracellular ions, metabolites, macromolecules, andorganelles. Classes of these reagents include labeling reagents thatmeasure the distribution and amount of molecules in living and fixedcells, environmental indicators to report signal transduction events intime and space, and fluorescent protein biosensors to measure targetmolecular activities within living cells. A multiparameter approach thatcombines several reagents in a single cell is a powerful new tool fordrug discovery.

[0112] The method of the present invention is based on the high affinityof fluorescent or luminescent molecules for specific cellularcomponents. The affinity for specific components is governed by physicalforces such as ionic interactions, covalent bonding (which includeschimeric fusion with protein-based chromophores, fluorophores, andlumiphores), as well as hydrophobic interactions, electrical potential,and, in some cases, simple entrapment within a cellular component. Theluminescent probes can be small molecules, labeled macromolecules, orgenetically engineered proteins, including, but not limited to greenfluorescent protein chimeras.

[0113] Those skilled in this art will recognize a wide variety offluorescent reporter molecules that can be used in the presentinvention, including, but not limited to, fluorescently labeledbiomolecules such as proteins, phospholipids and DNA hybridizing probes.Similarly, fluorescent reagents specifically synthesized with particularchemical properties of binding or association have been used asfluorescent reporter molecules (Barak et al., (1997), J. Biol. Chem.272:27497-27500; Southwick et al., (1990), Cytometry 11:418-430; Tsien(1989) in Methods in Cell Biology, Vol. 29 Taylor and Wang (eds.), pp.127-156). Fluorescently labeled antibodies are particularly usefulreporter molecules due to their high degree of specificity for attachingto a single molecular target in a mixture of molecules as complex as acell or tissue.

[0114] The luminescent probes can be synthesized within the living cellor can be transported into the cell via several non-mechanical modesincluding diffusion, facilitated or active transport,signal-sequence-mediated transport, and endocytotic or pinocytoticuptake. Mechanical bulk loading methods, which are well known in theart, can also be used to load luminescent probes into living cells(Barber et al. (1996), Neuroscience Letters 207:17-20; Bright et al.(1996), Cytometry 24:226-233; McNeil (1989) in Methods in Cell Biology,Vol. 29, Taylor and Wang (eds.), pp. 153-173). These methods includeelectroporation and other mechanical methods such as scrape-loading,bead-loading, impact-loading, syringe-loading, hypertonic and hypotonicloading. Additionally, cells can be genetically engineered to expressreporter molecules, such as GFP, coupled to a protein of interest aspreviously described (Chalfie and Prasher U.S. Pat. No. 5,491,084;Cubitt et al. (1995), Trends in Biochemical Science 20:448-455).

[0115] Once in the cell, the luminescent probes accumulate at theirtarget domain as a result of specific and high affinity interactionswith the target domain or other modes of molecular targeting such assignal-sequence-mediated transport. Fluorescently labeled reportermolecules are useful for determining the location, amount and chemicalenvironment of the reporter. For example, whether the reporter is in alipophilic membrane environment or in a more aqueous environment can bedetermined (Giuliano et al. (1995), Ann. Rev. of Biophysics andBiomolecular Structure 24:405-434; Giuliano and Taylor (1995), Methodsin Neuroscience 27.1-16). The pH environment of the reporter can bedetermined (Bright et al. (1989), J. Cell Biology 104:1019-1033;Giuliano et al. (1987), Anal. Biochem. 167:362-371; Thomas et al.(1979), Biochemistry 18:2210-2218). It can be determined whether areporter having a chelating group is bound to an ion, such as Ca++, ornot (Bright et al. (1989), In Methods in Cell Biology, Vol. 30, Taylorand Wang (eds.), pp. 157-192; Shimoura et al. (1988), J. of Biochemistry(Tokyo) 251:405-410; Tsien (1989) In Methods in Cell Biology, Vol. 30,Taylor and Wang (eds.), pp. 127-156).

[0116] Furthermore, certain cell types within an organism may containcomponents that can be specifically labeled that may not occur in othercell types. For example, epithelial cells often contain polarizedmembrane components. That is, these cells asymmetrically distributemacromolecules along their plasma membrane. Connective or supportingtissue cells often contain granules in which are trapped moleculesspecific to that cell type (e.g., heparin, histamine, serotonin, etc.).Most muscular tissue cells contain a sarcoplasmic reticulum, aspecialized organelle whose function is to regulate the concentration ofcalcium ions within the cell cytoplasm. Many nervous tissue cellscontain secretory granules and vesicles in which are trappedneurohormones or neurotransmitters. Therefore, fluorescent molecules canbe designed to label not only specific components within specific cells,but also specific cells within a population of mixed cell types.

[0117] Those skilled in the art will recognize a wide variety of ways tomeasure fluorescence. For example, some fluorescent reporter moleculesexhibit a change in excitation or emission spectra, some exhibitresonance energy transfer where one fluorescent reporter losesfluorescence, while a second gains in fluorescence, some exhibit a loss(quenching) or appearance of fluorescence, while some report rotationalmovements (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomol.Structure 24:405-434; Giuliano et al. (1995), Methods in Neuroscience27:1-16).

[0118] Scanning Cell Arrays

[0119] Referring to FIG. 9, a preferred embodiment is provided toanalyze cells that comprises operator-directed parameters being selectedbased on the assay being conducted, data acquisition by the cellscreening system on the distribution of fluorescent signals within asample, and interactive data review and analysis. At the start of anautomated scan the operator enters information 100 that describes thesample, specifies the filter settings and fluorescent channels to matchthe biological labels being used and the information sought, and thenadjusts the camera settings to match the sample brightness. Forflexibility to handle a range of samples, the software allows selectionof various parameter settings used to identify nuclei and cytoplasm, andselection of different fluorescent reagents, identification of cells ofinterest based on morphology or brightness, and cell numbers to beanalyzed per well. These parameters are stored in the system's for easyretrieval for each automated run. The system's interactive cellidentification mode simplifies the selection of morphological parameterlimits such as the range of size, shape, and intensity of cells to beanalyzed. The user specifies which wells of the plate the system willscan and how many fields or how many cells to analyze in each well.Depending on the setup mode selected by the user at step 101, the systemeither automatically pre-focuses the region of the plate to be scannedusing an autofocus procedure to “find focus” of the plate 102 or theuser interactively pre-focuses 103 the scanning region by selectingthree “tag” points which define the rectangular area to be scanned. Aleast-squares fit “focal plane model” is then calculated from these tagpoints to estimate the focus of each well during an automated scan. Thefocus of each well is estimated by interpolating from the focal planemodel during a scan.

[0120] During an automated scan, the software dynamically displays thescan status, including the number of cells analyzed, the current wellbeing analyzed, images of each independent wavelength as they areacquired, and the result of the screen for each well as it isdetermined. The plate 4 (FIG. 1) is scanned in a serpentine style as thesoftware automatically moves the motorized microscope XY stage 3 fromwell to well and field to field within each well of a 96-well plate.Those skilled in the programming art will recognize how to adaptsoftware for scanning of other microplate formats such as 24, 48, and384 well plates. The scan pattern of the entire plate as well as thescan pattern of fields within each well are programmed. The systemadjusts sample focus with an autofocus procedure 104 (FIG. 9) throughthe Z axis focus drive 5, controls filter selection via a motorizedfilter wheel 19, and acquires and analyzes images of up to fourdifferent colors (“channels” or “wavelengths”).

[0121] The autofocus procedure is called at a user selected frequency,typically for the first field in each well and then once every 4 to 5fields within each well. The autofocus procedure calculates the startingZ-axis point by interpolating from the pre-calculated plane focal model.Starting a programmable distance above or below this set point, theprocedure moves the mechanical Z-axis through a number of differentpositions, acquires an image at each position, and finds the maximum ofa calculated focus score that estimates the contrast of each image. TheZ position of the image with the maximum focus score determines the bestfocus for a particular field. Those skilled in the art will recognizethis as a variant of automatic focusing methods as described in Harms etal. in Cytometry 5 (1984), 236-243, Groen et al. in Cytometry 6 (1985),81-91, and Firestone et al. in Cytometry 12 (1991), 195-206.

[0122] For image acquisition, the camera's exposure time is separatelyadjusted for each dye to ensure a high-quality image from each channel.Software procedures can be called, at the user's option, to correct forregistration shifts between wavelengths by accounting for linear (X andY) shifts between wavelengths before making any further measurements.The electronic shutter 18 is controlled so that sample photo-bleachingis kept to a minimum. Background shading and uneven illumination can becorrected by the software using methods known in the art (Bright et al.(1987), J. Cell Biol. 104:1019-1033).

[0123] In one channel, images are acquired of a primary marker 105 (FIG.9) (typically cell nuclei counterstained with DAPI or PI fluorescentdyes) which are segmented (“identified”) using an adaptive thresholdingprocedure. The adaptive thresholding procedure 106 is used todynamically select the threshold of an image for separating cells fromthe background. The staining of cells with fluorescent dyes can vary toan unknown degree across cells in a microtiter plate sample as well aswithin images of a field of cells within each well of a microtiterplate. This variation can occur as a result of sample preparation and/orthe dynamic nature of cells. A global threshold is calculated for thecomplete image to separate the cells from background and account forfield to field variation. These global adaptive techniques are variantsof those described in the art. (Kittler et al. in Computer Vision,Graphics, and Image Processing 30 (1985), 125-147, Ridler et al. in IEEETrans. Systems, Man, and Cybernetics (1978), 630-632.)

[0124] An alternative adaptive thresholding method utilizes local regionthresholding in contrast to global image thresholding. Image analysis oflocal regions leads to better overall segmentation since staining ofcell nuclei (as well as other labeled components) can vary across animage. Using this global/local procedure, a reduced resolution image(reduced in size by a factor of 2 to 4) is first globally segmented(using adaptive thresholding) to find regions of interest in the image.These regions then serve as guides to more fully analyze the sameregions at full resolution. A more localized threshold is thencalculated (again using adaptive thresholding) for each region ofinterest.

[0125] The output of the segmentation procedure is a binary imagewherein the objects are white and the background is black. This binaryimage, also called a mask in the art, is used to determine if the fieldcontains objects 107. The mask is labeled with a blob labeling methodwhereby each object (or blob) has a unique number assigned to it.Morphological features, such as area and shape, of the blobs are used todifferentiate blobs likely to be cells from those that are consideredartifacts. The user pre-sets the morphological selection criteria byeither typing in known cell morphological features or by using theinteractive training utility. If objects of interest are found in thefield, images are acquired for all other active channels 108, otherwisethe stage is advanced to the next field 109 in the current well. Eachobject of interest is located in the image for further analysis 110. Thesoftware determines if the object meets the criteria for a valid cellnucleus 111 by measuring its morphological features (size and shape).For each valid cell, the XYZ stage location is recorded, a small imageof the cell is stored, and features are measured 112.

[0126] The cell scanning method of the present invention can be used toperform many different assays on cellular samples by applying a numberof analytical methods simultaneously to measure features at multiplewavelengths. An example of one such assay provides for the followingmeasurements:

[0127] 1. The total fluorescent intensity within the cell nucleus forcolors 1-4

[0128] 2. The area of the cell nucleus for color 1 (the primary marker)

[0129] 3. The shape of the cell nucleus for color 1 is described bythree shape features:

[0130] a) perimeter squared area

[0131] b) box area ratio

[0132] c) height width ratio

[0133] 4. The average fluorescent intensity within the cell nucleus forcolors 1-4 (i.e. #1 divided by #2)

[0134] 5. The total fluorescent intensity of a ring outside the nucleus(see FIG. 10) that represents fluorescence of the cell's cytoplasm(cytoplasmic mask) for colors 2-4

[0135] 6. The area of the cytoplasmic mask

[0136] 7. The average fluorescent intensity of the cytoplasmic mask forcolors 2-4 (i.e. #5 divided by #6)

[0137] 8. The ratio of the average fluorescent intensity of thecytoplasmic mask to average fluorescent intensity within the cellnucleus for colors 2-4 (i.e. #7 divided by #4)

[0138] 9. The difference of the average fluorescent intensity of thecytoplasmic mask and the average fluorescent intensity within the cellnucleus for colors 2-4 (i.e. #7 minus #4)

[0139] 10. The number of fluorescent domains (also call spots, dots, orgrains) within the cell nucleus for colors 2-4

[0140] Features 1 through 4 are general features of the different cellscreening assays of the invention. These steps are commonly used in avariety of image analysis applications and are well known in art (Russ(1992) The Image Processing Handbook, CRC Press Inc.; Gonzales et al.(1987), Digital Image Processing. Addison-Wesley Publishing Co. pp.391-448). Features 5-9 have been developed specifically to providemeasurements of a cell's fluorescent molecules within the localcytoplasmic region of the cell and the translocation (i.e. movement) offluorescent molecules from the cytoplasm to the nucleus. These features(steps 5-9) are used for analyzing cells in microplates for theinhibition of nuclear translocation. For example, inhibition of nucleartranslocation of transcription factors provides a novel approach toscreening intact cells (detailed examples of other types of screens willbe provided below). A specific method measures the amount of probe inthe nuclear region (feature 4) versus the local cytoplasmic region(feature 7) of each cell. Quantification of the difference between thesetwo sub-cellular compartments provides a measure of cytoplasm-nucleartranslocation (feature 9).

[0141] Feature 10 describes a screen used for counting of DNA or RNAprobes within the nuclear region in colors 2-4. For example, probes arecommercially available for identifying chromosome-specific DNA sequences(Life Technologies, Gaithersburg, Md.; Genosys, Woodlands, Tex.;Biotechnologies, Inc., Richmond, Calif.; Bio 101, Inc., Vista, Calif.)Cells are three-dimensional in nature and when examined at a highmagnification under a microscope one probe may be in-focus while anothermay be completely out-of-focus. The cell screening method of the presentinvention provides for detecting three-dimensional probes in nuclei byacquiring images from multiple focal planes. The software moves theZ-axis motor drive 5 (FIG. 1) in small steps where the step distance isuser selected to account for a wide range of different nucleardiameters. At each of the focal steps, an image is acquired. The maximumgray-level intensity from each pixel in each image is found and storedin a resulting maximum projection image. The maximum projection image isthen used to count the probes. The above method works well in countingprobes that are not stacked directly above or below another one. Toaccount for probes stacked on top of each other in the Z-direction,users can select an option to analyze probes in each of the focal planesacquired. In this mode, the scanning system performs the maximum planeprojection method as discussed above, detects probe regions of interestin this image, then further analyzes these regions in all the focalplane images.

[0142] After measuring cell features 112 (FIG. 9), the system checks ifthere are any unprocessed objects in the current field 113. If there areany unprocessed objects, it locates the next object 110 and determineswhether it meets the criteria for a valid cell nucleus 111, and measuresits features. Once all the objects in the current field are processed,the system determines whether analysis of the current plate is complete114; if not, it determines the need to find more cells in the currentwell 115. If the need exists, the system advances the XYZ stage to thenext field within the current well 109 or advances the stage to the nextwell 116 of the plate.

[0143] After a plate scan is complete, images and data can be reviewedwith the system's image review, data review, and summary reviewfacilities. All images, data, and settings from a scan are archived inthe system's database for later review or for interfacing with a networkinformation management system. Data can also be exported to otherthird-party statistical packages to tabulate results and generate otherreports. Users can review the images alone of every cell analyzed by thesystem with an interactive image review procedure 117. The user canreview data on a cell-by-cell basis using a combination of interactivegraphs, a data spreadsheet of measured features, and images of all thefluorescence channels of a cell of interest with the interactivecell-by-cell data review procedure 118. Graphical plotting capabilitiesare provided in which data can be analyzed via interactive graphs suchas histograms and scatter plots. Users can review summary data that areaccumulated and summarized for all cells within each well of a platewith an interactive well-by-well data review procedure 119. Hard copiesof graphs and images can be printed on a wide range of standardprinters.

[0144] As a final phase of a complete scan, reports can be generated onone or more statistics of the measured features. Users can generate agraphical report of data summarized on a well-by-well basis for thescanned region of the plate using an interactive report generationprocedure 120. This report includes a summary of the statistics by wellin tabular and graphical format and identification information on thesample. The report window allows the operator to enter comments aboutthe scan for later retrieval. Multiple reports can be generated on manystatistics and be printed with the touch of one button. Reports can bepreviewed for placement and data before being printed.

[0145] The above-recited embodiment of the method operates in a singlehigh resolution mode referred to as the high content screening (HCS)mode. The HCS mode provides sufficient spatial resolution within a well(on the order of 1 μm) to define the distribution of material within thewell, as well as within individual cells in the well. The high degree ofinformation content accessible in that mode, comes at the expense ofspeed and complexity of the required signal processing.

[0146] In an alternative embodiment, a high throughput system (HTS) isdirectly coupled with the HCS either on the same platform or on twoseparate platforms connected electronically (e.g. via a local areanetwork). This embodiment of the invention, referred to as a dual modeoptical system, has the advantage of increasing the throughput of an HCSby coupling it with an HTS and thereby requiring slower high resolutiondata acquisition and analysis only on the small subset of wells thatshow a response in the coupled HTS. . High throughput ‘whole plate’reader systems are well known in the art and are commonly used as acomponent of an HTS system used to screen large numbers of compounds(Beggs et al. (1997), supra; McCaffrey et al. (1996), supra). The HTS ofthe present invention is carried out on the microtiter plate ormicrowell array by reading many or all wells in the plate simultaneouslywith sufficient resolution to make determinations on a well-by-wellbasis. That is, calculations are made by averaging the total signaloutput of many or all the cells or the bulk of the material in eachwell. Wells that exhibit some defined response in the HTS (the ‘hits’)are flagged by the system. Then on the same microtiter plate ormicrowell array, each well identified as a hit is measured via HCS asdescribed above. Thus, the dual mode process involves:

[0147] 1. Rapidly measuring numerous wells of a microtiter plate ormicrowell array,

[0148] 2. Interpreting the data to determine the overall activity offluorescently labeled reporter molecules in the cells on a well-by-wellbasis to identify “hits” (wells that exhibit a defined response),

[0149] 3. Imaging numerous cells in each “hit” well, and

[0150] 4. Interpreting the digital image data to determine thedistribution, environment or activity of the fluorescently labeledreporter molecules in the individual cells (i.e. intracellularmeasurements) and the distribution of the cells to test for specificbiological functions

[0151] In a preferred embodiment of dual mode processing (FIG. 11), atthe start of a run 301, the operator enters information 302 thatdescribes the plate and its contents, specifies the filter settings andfluorescent channels to match the biological labels being used, theinformation sought and the camera settings to match the samplebrightness. These parameters are stored in the system's database foreasy retrieval for each automated run. The microtiter plate or microwellarray is loaded into the cell screening system 303 either manually orautomatically by controlling a robotic loading device. An optionalenvironmental chamber 304 is controlled by the system to maintain thetemperature, humidity and CO₂ levels in the air surrounding live cellsin the microtiter plate or microwell array. An optional fluid deliverydevice 305 (see FIG. 8) is controlled by the system to dispense fluidsinto the wells during the scan.

[0152] High throughput processing 306 is first performed on themicrotiter plate or microwell array by acquiring and analyzing thesignal from each of the wells in the plate. The processing performed inhigh throughput mode 307 is illustrated in FIG. 12 and described below.Wells that exhibit some selected intensity response in this highthroughput mode (“hits”) are identified by the system. The systemperforms a conditional operation 308 that tests for hits. If hits arefound, those specific hit wells are further analyzed in high content(micro level) mode 309. The processing performed in high content mode312 is illustrated in FIG. 13. The system then updates 310 theinformatics database 311 with results of the measurements on the plate.If there are more plates to be analyzed 313 the system loads the nextplate 303; otherwise the analysis of the plates terminates 314.

[0153] The following discussion describes the high throughput modeillustrated in FIG. 12. The preferred embodiment of the system, thesingle platform dual mode screening system, will be described. Thoseskilled in the art will recognize that operationally the dual platformsystem simply involves moving the plate between two optical systemsrather than moving the optics. Once the system has been set up and theplate loaded, the system begins the HTS acquisition and analysis 401.The HTS optical module is selected by controlling a motorized opticalpositioning device 402 on the dual mode system. In one fluorescencechannel, data from a primary marker on the plate is acquired 403 andwells are isolated from the plate background using a masking procedure404. Images are also acquired in other fluorescence channels being used405. The region in each image corresponding to each well 406 is measured407. A feature calculated from the measurements for a particular well iscompared with a predefined threshold or intensity response 408, andbased on the result the well is either flagged as a “hit” 409 or not.The locations of the wells flagged as hits are recorded for subsequenthigh content mode processing. If there are wells remaining to beprocessed 410 the program loops back 406 until all the wells have beenprocessed 411 and the system exits high throughput mode.

[0154] Following HTS analysis, the system starts the high content modeprocessing 501 defined in FIG. 13. The system selects the HCS opticalmodule 502 by controlling the motorized positioning system. For each“hit” well identified in high throughput mode, the XY stage location ofthe well is retrieved from memory or disk and the stage is then moved tothe selected stage location 503. The autofocus procedure 504 is calledfor the first field in each hit well and then once every 5 to 8 fieldswithin each well. In one channel, images are acquired of the primarymarker 505 (typically cell nuclei counterstained with DAPI, Hoechst orPI fluorescent dye). The images are then segmented (separated intoregions of nuclei and non-nuclei) using an adaptive thresholdingprocedure 506. The output of the segmentation procedure is a binary maskwherein the objects are white and the background is black. This binaryimage, also called a mask in the art, is used to determine if the fieldcontains objects 507. The mask is labeled with a blob labeling methodwhereby each object (or blob) has a unique number assigned to it. Ifobjects are found in the field, images are acquired for all other activechannels 508, otherwise the stage is advanced to the next field 514 inthe current well. Each object is located in the image for furtheranalysis 509. Morphological features, such as area and shape of theobjects, are used to select objects likely to be cell nuclei 510, anddiscard (do no further processing on) those that are consideredartifacts. For each valid cell nucleus, the XYZ stage location isrecorded, a small image of the cell is stored, and assay specificfeatures are measured 511. The system then performs multiple tests onthe cells by applying several analytical methods to measure features ateach of several wavelengths. After measuring the cell features, thesystems checks if there are any unprocessed objects in the current field512. If there are any unprocessed objects, it locates the next object509 and determines whether it meets the criteria for a valid cellnucleus 510, and measures its features. After processing all the objectsin the current field, the system deteremines whether it needs to findmore cells or fields in the current well 513. If it needs to find morecells or fields in the current well it advances the XYZ stage to thenext field within the current well 515. Otherwise, the system checkswhether it has any remaining hit wells to measure 515. If so, itadvances to the next hit well 503 and proceeds through another cycle ofacquisition and analysis, otherwise the HCS mode is finished 516.

[0155] In an alternative embodiment of the present invention, a methodof kinetic live cell screening is provided. The previously describedembodiments of the invention are used to characterize the spatialdistribution of cellular components at a specific point in time, thetime of chemical fixation. As such, these embodiments have limitedutility for implementing kinetic based screens, due to the sequentialnature of the image acquisition, and the amount of time required to readall the wells on a plate. For example, since a plate can require 30-60minutes to read through all the wells, only very slow kinetic processescan be measured by simply preparing a plate of live cells and thenreading through all the wells more than once. Faster kinetic processescan be measured by taking multiple readings of each well beforeproceeding to the next well, but the elapsed time between the first andlast well would be too long, and fast kinetic processes would likely becomplete before reaching the last well.

[0156] The kinetic live cell extension of the invention enables thedesign and use of screens in which a biological process is characterizedby its kinetics instead of, or in addition to, its spatialcharacteristics. In many cases, a response in live cells can be measuredby adding a reagent to a specific well and making multiple measurementson that well with the appropriate timing. This dynamic live cellembodiment of the invention therefore includes apparatus for fluiddelivery to individual wells of the system in order to deliver reagentsto each well at a specific time in advance of reading the well. Thisembodiment thereby allows kinetic measurements to be made with temporalresolution of seconds to minutes on each well of the plate. To improvethe overall efficiency of the dynamic live cell system, the acquisitioncontrol program is modified to allow repetitive data collection fromsub-regions of the plate, allowing the system to read other wellsbetween the time points required for an individual well.

[0157]FIG. 8 describes an example of a fluid delivery device for usewith the live cell embodiment of the invention and is described above.This set-up allows one set of pipette tips 705, or even a single pipettetip, to deliver reagent to all the wells on the plate. The bank ofsyringe pumps 701 can be used to deliver fluid to 12 wellssimultaneously, or to fewer wells by removing some of the tips 705. Thetemporal resolution of the system can therefore be adjusted, withoutsacrificing data collection efficiency, by changing the number of tipsand the scan pattern as follows. Typically, the data collection andanalysis from a single well takes about 5 seconds. Moving from well towell and focusing in a well requires about 5 seconds, so the overallcycle time for a well is about 10 seconds. Therefore, if a singlepipette tip is used to deliver fluid to a single well, and data iscollected repetitively from that well, measurements can be made withabout 5 seconds temporal resolution. If 6 pipette tips are used todeliver fluids to 6 wells simultaneously, and the system repetitivelyscans all 6 wells, each scan will require 60 seconds, therebyestablishing the temporal resolution. For slower processes which onlyrequire data collection every 8 minutes, fluids can be delivered to onehalf of the plate, by moving the plate during the fluid delivery phase,and then repetitively scanning that half of the plate. Therefore, byadjusting the size of the sub-region being scanned on the plate, thetemporal resolution can be adjusted without having to insert wait timesbetween acquisitions. Because the system is continuously scanning andacquiring data, the overall time to collect a kinetic data set from theplate is then simply the time to perform a single scan of the plate,multiplied by the number of time points required. Typically, 1 timepoint before addition of compounds and 2 or 3 time points followingaddition should be sufficient for screening purposes.

[0158]FIG. 14 shows the acquisition sequence used for kinetic analysis.The start of processing 801 is configuration of the system, much ofwhich is identical to the standard HCS configuration. In addition, theoperator must enter information specific to the kinetic analysis beingperformed 802, such as the sub-region size, the number of time pointsrequired, and the required time increment. A sub-region is a group ofwells that will be scanned repetitively in order to accumulate kineticdata. The size of the sub-region is adjusted so that the system can scana whole sub-region once during a single time increment, thus minimizingwait times. The optimum sub-region size is calculated from the setupparameters, and adjusted if necessary by the operator. The system thenmoves the plate to the first sub-region 803, and to the first well inthat sub-region 804 to acquire the prestimulation (time=0) time points.The acquisition sequence performed in each well is exactly the same asthat required for the specific HCS being run in kinetic mode. FIG. 15details a flow chart for that processing. All of the steps between thestart 901 and the return 902 are identical to those described as steps504-514 in FIG. 13.

[0159] After processing each well in a sub-region, the system checks tosee if all the wells in the sub-region have been processed 806 (FIG.14), and cycles through all the wells until the whole region has beenprocessed. The system then moves the plate into position for fluidaddition, and controls fluidic system delivery of fluids to the entiresub-region 807. This may require multiple additions for sub-regionswhich span several rows on the plate, with the system moving the plateon the X,Y stage between additions. Once the fluids have been added, thesystem moves to the first well in the sub-region 808 to beginacquisition of time points. The data is acquired from each well 809 andas before the system cycles through all the wells in the sub-region 810.After each pass through the sub-region, the system checks whether allthe time points have been collected 811 and if not, pauses 813 ifnecessary 812 to stay synchronized with the requested time increment.Otherwise, the system checks for additional sub-regions on the plate 814and either moves to the next sub-region 803 or finishes 815. Thus, thekinetic analysis mode comprises operator identification of sub-regionsof the microtiter plate or microwells to be screened, based on thekinetic response to be investigated, with data acquisitions within asub-region prior to data acquisition in subsequent sub-regions.

[0160] Specific Screens

[0161] In another aspect of the present invention, cell screeningmethods and machine readable storage medium comprising a programcontaining a set of instructions for causing a cell screening system toexecute procedures for defining the distribution and activity ofspecific cellular constituents and processes is provided. In a preferredembodiment, the cell screening system comprises a high magnificationfluorescence optical system with a stage adapted for holding cells and ameans for moving the stage, a digital camera, a light source forreceiving and processing the digital data from the digital camera, and acomputer means for receiving and processing the digital data from thedigital camera. This aspect of the invention comprises programs thatinstruct the cell screening system to define the distribution andactivity of specific cellular constituents and processes, using theluminescent probes, the optical imaging system, and the patternrecognition software of the invention. Preferred embodiments of themachine readable storage medium comprise programs consisting of a set ofinstructions for causing a cell screening system to execute theprocedures set forth in FIGS. 9, 11, 12, 13, 14 or 15. Another preferredembodiment comprises a program consisting of a set of instructions forcausing a cell screening system to execute procedures for detecting thedistribution and activity of specific cellular constituents andprocesses. In most preferred embodiments, the cellular processesinclude, but are not limited to, nuclear translocation of a protein,cellular morphology, apoptosis, receptor internalization, andprotease-induced translocation of a protein.

[0162] In a preferred embodiment, the cell screening methods are used toidentify compounds that modify the various cellular processes. The cellscan be contacted with a test compound, and the effect of the testcompound on a particular cellular process can be analyzed.Alternatively, the cells can be contacted with a test compound and aknown agent that modifies the particular cellular process, to determinewhether the test compound can inhibit or enhance the effect of the knownagent. Thus, the methods can be used to identify test compounds thatincrease or decrease a particular cellular response, as well as toidentify test compounds that affects the ability of other agents toincrease or decrease a particular cellular response.

[0163] In another preferred embodiment, the locations containing cellsare analyzed using the above methods at low resolution in a highthroughput mode, and only a subset of the locations containing cells areanalyzed in a high content mode to obtain luminescent signals from theluminescently labeled reporter molecules in subcellular compartments ofthe cells being analyzed.

[0164] The following examples are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention, asdefined in the claims appended hereto.

[0165] The various chemical compounds, reagents, dyes, and antibodiesthat are referred to in the following Examples are commerciallyavailable from such sources as Sigma Chemical (St. Louis, Mo.),Molecular Probes (Eugene, Oreg.), Aldrich Chemical Company (Milwaukee,Wis.), Accurate Chemical Company (Westbury, N.Y.), Jackson Immunolabs,and Clontech (Palo Alto, Calif.).

EXAMPLE 1 Cytoplasm to Nucleus Translocation Screening

[0166] a. Transcription Factors

[0167] Regulation of transcription of some genes involves activation ofa transcription factor in the cytoplasm, resulting in that factor beingtransported into the nucleus where it can initiate transcription of aparticular gene or genes. This change in transcription factordistribution is the basis of a screen for the cell-based screeningsystem to detect compounds that inhibit or induce transcription of aparticular gene or group of genes. A general description of the screenis given followed by a specific example.

[0168] The distribution of the transcription factor is determined bylabeling the nuclei with a DNA specific fluorophore like Hoechst 33423and the transcription factor with a specific fluorescent antibody. Afterautofocusing on the Hoechst labeled nuclei, an image of the nuclei isacquired in the cell-based screening system and used to create a mask byone of several optional thresholding methods, as described supra. Themorphological descriptors of the regions defined by the mask arecompared with the user defined parameters and valid nuclear masks areidentified and used with the following method to extract transcriptionfactor distributions. Each valid nuclear mask is eroded to define aslightly smaller nuclear region. The original nuclear mask is thendilated in two steps to define a ring shaped region around the nucleus,which represents a cytoplasmic region. The average antibody fluorescencein each of these two regions is determined, and the difference betweenthese averages is defined as the NucCyt Difference. Two examples ofdetermining nuclear translocation are discussed below and illustrated inFIGS. 10A-J. FIG. 10A illustrates an unstimulated cell with its nucleus200 labeled with a blue fluorophore and a transcription factor in thecytoplasm 201 labeled with a green fluorophore. FIG. 10B illustrates thenuclear mask 202 derived by the cell-based screening system. FIG. 10Cillustrates the cytoplasm 203 of the unstimulated cell imaged at a greenwavelength. FIG. 10D illustrates the nuclear mask 202 is eroded(reduced) once to define a nuclear sampling region 204 with minimalcytoplasmic distribution. The nucleus boundary 202 is dilated (expanded)several times to form a ring that is 2-3 pixels wide that is used todefine the cytoplasmic sampling region 205 for the same cell. FIG. 10Efurther illustrates a side view which shows the nuclear sampling region204 and the cytoplasmic sampling region 205. Using these two samplingregions, data on nuclear translocation can be automatically analyzed bythe cell-based screening system on a cell by cell basis. FIGS. 10F-Jillustrates the strategy for determining nuclear translocation in astimulated cell. FIG. 10F illustrates a stimulated cell with its nucleus206 labeled with a blue fluorophore and a transcription factor in thecytoplasm 207 labeled with a green fluorophore. The nuclear mask 208 inFIG. 10G is derived by the cell based screening system. FIG. 10Hillustrates the cytoplasm 209 of a stimulated cell imaged at a greenwavelength. FIG. 10I illustrates the nuclear sampling region 211 andcytoplasmic sampling region 212 of the stimulated cell. FIG. 10J furtherillustrates a side view which shows the nuclear sampling region 211 andthe cytoplasmic sampling region 212.

[0169] A specific application of this method has been used to validatethis method as a screen. A human cell line was plated in 96 wellmicrotiter plates. Some rows of wells were titrated with IL-1, a knowninducer of the NF-KB transcription factor. The cells were then fixed andstained by standard methods with a fluorescein labeled antibody to thetranscription factor, and Hoechst 33423. The cell-based screening systemwas used to acquire and analyze images from this plate and the NucCytDifference was found to be strongly correlated with the amount ofagonist added to the wells as illustrated in FIG. 16. In a secondexperiment, an antagonist to the receptor for IL-1, IL-1RA was titratedin the presence of IL-1α, progressively inhibiting the translocationinduced by IL-1α. The NucCyt Difference was found to strongly correlatewith this inhibition of translocation, as illustrated in FIG. 17.

[0170] Additional experiments have shown that the NucCyt Difference, aswell as the NucCyt ratio, gives consistent results over a wide range ofcell densities and reagent concentrations, and can therefore beroutinely used to screen compound libraries for specific nucleartranslocation activity. Furthermore, the same method can be used withantibodies to other transcription factors, or GFP-transcription factorchimeras, or fluorescently labeled transcription factors introduced intoliving or fixed cells, to screen for effects on the regulation oftranscription factor activity.

[0171]FIG. 18 is a representative display on a PC screen of data whichwas obtained in accordance with Example 1. Graph 1 180 plots thedifference between the average antibody fluorescence in the nuclearsampling region and cytoplasmic sampling region, NucCyt Differenceverses Well #. Graph 2 181 plots the average fluorescence of theantibody in the nuclear sampling region, NP1 average, versus the Well #.Graph 3 182 plots the average antibody fluorescence in the cytoplasmicsampling region, LIP1 average, versus Well #. The software permitsdisplaying data from each cell. For example, FIG. 18 shows a screendisplay 183, the nuclear image 184, and the fluorescent antibody image185 for cell #26.

[0172] NucCyt Difference referred to in graph 1 180 of FIG. 18 is thedifference between the average cytoplasmic probe (fluorescent reportermolecule) intensity and the average nuclear probe (fluorescent reportermolecule) intensity. NP1 average referred to in graph 2 181 of FIG. 18is the average of cytoplasmic probe (fluorescent reporter molecule)intensity within the nuclear sampling region. LIP1 average referred toin graph 3 182 of FIG. 18 is the average probe (fluorescent reportermolecule) intensity within the cytoplasmic sampling region.

[0173] It will be understood by one of skill in the art that this aspectof the invention can be performed using other transcription factors thattranslocate from the cytoplasm to the nucleus upon activation. Inanother specific example, activation of the c-fos transcription factorwas assessed by defining its spatial position within cells. Activatedc-fos is found only within the nucleus, while inactivated c-fos resideswithin the cytoplasm.

[0174] 3T3 cells were plated at 5000-10000 cells per well in aPolyfiltronics 96-well plate. The cells were allowed to attach and growovernight. The cells were rinsed twice with 100 μl serum-free medium,incubated for 24-30 hours in serum-free MEM culture medium, and thenstimulated with platelet derived growth factor (PDGF-BB) (Sigma ChemicalCo., St. Louis, Mo.) diluted directly into serum free medium atconcentrations ranging from 1-50 ng/ml for an average time of 20minutes.

[0175] Following stimulation, cells were fixed for 20 minutes in 3.7%formaldehyde solution in 1×Hanks buffered saline solution (HBSS). Afterfixation, the cells were washed with HBSS to remove residual fixative,permeabilized for 90 seconds with 0.5% Triton X-100 solution in HBSS,and washed twice with HBSS to remove residual detergent. The cells werethen blocked for 15 minutes with a 0.1% solution of BSA in HBSS, andfurther washed with HBSS prior to addition of diluted primary antibodysolution.

[0176] c-Fos rabbit polyclonal antibody (Calbiochem, PC05) was diluted1:50 in HBSS, and 50 μl of the dilution was applied to each well. Cellswere incubated in the presence of primary antibody for one hour at roomtemperature, and then incubated for one hour at room temperature in alight tight container with goat anti-rabbit secondary antibodyconjugated to ALEXA™ 488 (Molecular Probes), diluted 1:500 from a 100μg/ml stock in HBSS. Hoechst DNA dye (Molecular Probes) was then addedat a 1:1000 dilution of the manufacturer's stock solution (10 mg/ml).The cells were then washed with HBSS, and the plate was sealed prior toanalysis with the cell screening system of the invention. The data fromthese experiments demonstrated that the methods of the invention couldbe used to measure transcriptional activation of c-fos by defining itsspatial position within cells.

[0177] One of skill in the art will recognize that while the followingmethod is applied to detection of c-fos activation, it can be applied tothe analysis of any transcription factor that translocates from thecytoplasm to the nucleus upon activation. Examples of such transcriptionfactors include, but are not limited to fos and jun homologs, NF-KB(nuclear factor kappa from B cells), NFAT (nuclear factor of activatedT-lymphocytes), and STATs (signal transducer and activator oftranscription) factors (For example, see Strehlow, I., and Schindler, C.1998. J. Biol. Chem. 273:28049-28056; Chow, et al. 1997 Science.278:1638-1641; Ding et al. 1998 J. Biol. Chem. 273:28897-28905; Baldwin,1996. Annu Rev Immunol. 14:649-83; Kuo, C. T., and J. M. Leiden. 1999.Annu Rev Immunol. 17:149-87; Rao, et al. 1997. Annu Rev Immunol.15:707-47; Masuda, et al. 1998. Cell Signal. 10:599-611; Hoey, T., andU. Schindler. 1998. Curr Opin Genet Dev. 8:582-7; Liu, et al. 1998. CurrOpin Immunol. 10:271-8.) Thus, in this aspect of the invention,indicator cells are treated with test compounds and the distribution ofluminescently labeled transcription factor is measured in space and timeusing a cell screening system, such as the one disclosed above. Theluminescently labeled transcription factor may be expressed by or addedto the cells either before, together with, or after contacting the cellswith a test compound. For example, the transcription factor may beexpressed as a luminescently labeled protein chimera by transfectedindicator cells. Alternatively, the luminescently labeled transcriptionfactor may be expressed, isolated, and bulk-loaded into the indicatorcells as described above, or the transcription factor may beluminescently labeled after isolation. As a further alternative, thetranscription factor is expressed by the indicator cell, which issubsequently contacted with a luminescent label, such as an antibody,that detects the transcription factor.

[0178] In a further aspect, kits are provided for analyzingtranscription factor activation, comprising an antibody thatspecifically recognizes a transcription factor of interest, andinstructions for using the antibody for carrying out the methodsdescribed above. In a preferred embodiment, the transcriptionfactor-specific antibody, or a secondary antibody that detects thetranscription factor antibody, is luminescently labeled. In furtherpreferred embodiments, the kit contains cells that express thetranscription factor of interest, and/or the kit contains a compoundthat is known to modify activation of the transcription factor ofinterest, including but not limited to platelet derived growth factor(PDGF) and serum, which both modify fos activation; and interleukin 1(IL-1) and tumor necrosis factor (TNF), which both modify NF-KBactivation.

[0179] In another embodiment, the kit comprises a recombinant expressionvector comprising a nucleic acid encoding a transcription factor ofinterest that translocates from the cytoplasm to the nucleus uponactivation, and instructions for using the expression vector to identifycompounds that modify transcription factor activation in a cell ofinterest. Alternatively, the kits contain a purified, luminescentlylabeled transcription factor. In a preferred embodiment, thetranscription factor is expressed as a fusion protein with a luminescentprotein, including but not limited to green fluorescent protein,luceriferase, or mutants or fragments thereof. In various preferredembodiments, the kit further contains cells that are transfected withthe expression vector, an antibody or fragment that specifically bind tothe transcription factor of interest, and/or a compound that is known tomodify activation of the transcription factor of interest (as above).

[0180] b. Protein Kinases

[0181] The cytoplasm to nucleus screening methods can also be used toanalyze the activation of any protein kinase that is present in aninactive state in the cytoplasm and is transported to the nucleus uponactivation, or that phosphorylates a substrate that translocates fromthe cytoplasm to the nucleus upon phosphorylation. Examples ofappropriate protein kinases include, but are not limited toextracellular signal-regulated protein kinases (ERKs), c-Junamino-terminal kinases (JNKs), Fos regulating protein kinases (FRKs),p38 mitogen activated protein kinase (p38MAPK), protein kinase A (PKA),and mitogen activated protein kinase kinases (MAPKKs). (For example, seeHall, et al. 1999. J. Biol. Chem. 274:376-83; Han, et al. 1995. Biochim.Biophys. Acta. 1265:224-227; Jaaro et al. 1997. Proc. Natl. Acad. Sci.U.S.A. 94:3742-3747; Taylor, et al. 1994. J. Biol. Chem. 269:308-318;Zhao, Q., and F. S. Lee. 1999. J. Biol. Chem. 274:8355-8; Paolilloet al.1999. J. Biol Chem. 274:6546-52; Coso et al. 1995. Cell 81:1137-1146;Tibbles, L. A., and J. R. Woodgett. 1999. Cell Mol Life Sci. 55:1230-54;Schaeffer, H. J., and M. J. Weber. 1999. Mol Cell Biol. 19:2435-44.)

[0182] Alternatively, protein kinase activity is assayed by monitoringtranslocation of a luminescently labeled protein kinase substrate fromthe cytoplasm to the nucleus after being phosphorylated by the proteinkinase of interest. In this embodiment, the substrate isnon-phosphorylated and cytoplasmic prior to phosphorylation, and istranslocated to the nucleus upon phosphorylation by the protein kinase.There is no requirement that the protein kinase itself translocates fromthe cytoplasm to the nucleus in this embodiment. Examples of suchsubstrates (and the corresponding protein kinase) include, but are notlimited to c-jun (JNK substrate); fos (FRK substrate), and p38 (p38 MAPKsubstrate).

[0183] Thus, in these embodiments, indicator cells are treated with testcompounds and the distribution of luminescently labeled protein kinaseor protein kinase substrate is measured in space and time using a cellscreening system, such as the one disclosed above. The luminescentlylabeled protein kinase or protein kinase substrate may be expressed byor added to the cells either before, together with, or after contactingthe cells with a test compound. For example, the protein kinase orprotein kinase substrate may be expressed as a luminescently labeledprotein chimera by transfected indicator cells. Alternatively, theluminescently labeled protein kinase or protein kinase substrate may beexpressed, isolated, and bulk-loaded into the indicator cells asdescribed above, or the protein kinase or protein kinase substrate maybe luminescently labeled after isolation. As a further alternative, theprotein kinase or protein kinase substrate is expressed by the indicatorcell, which is subsequently contacted with a luminescent label, such asa labeled antibody, that detects the protein kinase or protein kinasesubstrate.

[0184] In a further embodiment, protein kinase activity is assayed bymonitoring the phosphorylation state (ie: phosphorylated or notphosphorylated) of a protein kinase substrate. In this embodiment, thereis no requirement that either the protein kinase or the protein kinasesubstrate translocate from the cytoplasm to the nucleus upon activation.In a preferred embodiment, phosphorylation state is monitored bycontacting the cells with an antibody that binds only to thephosphorylated form of the protein kinase substrate of interest (Forexample, as disclosed in U.S. Pat. No. 5,599,681).

[0185] In another preferred embodiment, a biosensor of phosphorylationis used. For example, a luminescently labeled protein or fragmentthereof can be fused to a protein that has been engineered to contain(a) a phosphorylation site that is recognized by a protein kinase ofinterest; and (b) a nuclear localization signal that is unmasked by thephosphorylation. Such a biosensor will thus be translocated to thenucleus upon phosphorylation, and its translocation can be used as ameasure of protein kinase activation.

[0186] In another aspect, kits are provided for analyzing protein kinaseactivation, comprising a primary antibody that specifically binds to aprotein kinase, a protein kinase substrate, or a phosphorylated form ofthe protein kinase substrate of interest and instructions for using theprimary antibody to identify compounds that modify protein kinaseactivation in a cell of interest. In a preferred embodiment, the primaryantibody, or a secondary antibody that detects the primary antibody, isluminescently labeled. In other preferred embodiments, the kit furthercomprises cells that express the protein kinase of interest, and/or acompound that is known to modify activation of the protein kinase ofinterest, including but not limited to dibutyryl cAMP (modifies PKA),forskolin (PKA), and anisomycin (p38MAPK).

[0187] Alternatively, the kits comprise an expression vector encoding aprotein kinase or a protein kinase substrate of interest thattranslocates from the cytoplasm to the nucleus upon activation andinstructions for using the expression vector to identify compounds thatmodify protein kinase activation in a cell of interest. Alternatively,the kits contain a purified, luminescently labeled protein kinase orprotein kinase substrate. In a preferred embodiment, the protein kinaseor protein kinase substrate of interest is expressed as a fusion proteinwith a luminescent protein. In further preferred embodiments, the kitfurther comprises cells that are transfected with the expression vector,an antibody or fragment thereof that specifically binds to the proteinkinase or protein kinase substrate of interest, and/or a compound thatis known to modify activation of the protein kinase of interest. (asabove)

[0188] In another aspect, the present invention comprises a machinereadable storage medium comprising a program containing a set ofinstructions for causing a cell screening system to execute the methodsdisclosed for analyzing transcription factor or protein kinaseactivation, wherein the cell screening system comprises an opticalsystem with a stage adapted for holding a plate containing cells, adigital camera, a means for directing fluorescence or luminescenceemitted from the cells to the digital camera, and a computer means forreceiving and processing the digital data from the digital camera.

EXAMPLE 2 Automated Screen for Compounds that Modify Cellular Morphology

[0189] Changes in cell size are associated with a number of cellularconditions, such as hypertrophy, cell attachment and spreading,differentiation, growth and division, necrotic and programmed celldeath, cell motility, morphogenesis, tube formation, and colonyformation.

[0190] For example, cellular hypertrophy has been associated with acascade of alterations in gene expression and can be characterized incell culture by an alteration in cell size, that is clearly visible inadherent cells growing on a coverslip.

[0191] Cell size can also be measured to determine the attachment andspreading of adherent cells. Cell spreading is the result of selectivebinding of cell surface receptors to substrate ligands and subsequentactivation of signaling pathways to the cytoskeleton. Cell attachmentand spreading to substrate molecules is an important step for themetastasis of cancer cells, leukocyte activation during the inflammatoryresponse, keratinocyte movement during wound healing, and endothelialcell movement during angiogenesis. Compounds that affect these surfacereceptors, signaling pathways, or the cytoskeleton will affect cellspreading and can be screened by measuring cell size.

[0192] Total cellular area can be monitored by labeling the entire cellbody or the cell cytoplasm using cytoskeletal markers, cytosolic volumemarkers, or cell surface markers, in conjunction with a DNA label.Examples of such labels (many available from Molecular Probes (Eugene,Oreg.) and Sigma Chemical Co. (St. Louis, Mo.)) include the following:CELL SIZE AND AREA MARKERS Cytoskeletal Markers ALEXA ™ 488 phalloidin(Molecular Probes, Oregon) Tubulin-green fluorescent protein chimerasCytokeratin-green fluorescent protein chimeras Antibodies tocytoskeletal proteins Cytosolic Volume Markers Green fluorescentproteins Chloromethylfluorescein diacetate (CMFDA) Calcein greenBCECF/AM ester Rhodamine dextran Cell Surface Markers for Lipid,Protein, or Oligosaccharide Dihexadecyl tetramethylindocarbocyanineperchlorate (DiIC16) lipid dyes Triethylammonium propyl dibutylaminostyryl pyridinium (FM 4-64, FM 1-43) lipid dyes MITOTRACKER ™ Green FMLectins to oligosaccarides such as fluorescein concanavalin A or wheatgerm agglutinin SYPRO ™ Red non-specific protein markers Antibodies tovarious surface proteins such as epidermal growth factor Biotin labelingof surface proteins followed by fluorescent strepavidin labeleing

[0193] Protocols for cell staining with these various agents are wellknown to those skilled in the art. Cells are stained live or afterfixation and the cell area can be measured. For example, live cellsstained with DiIC16 have homogeneously labeled plasma membranes, and theprojected cross-sectional area of the cell is uniformly discriminatedfrom background by fluorescence intensity of the dye. Live cells stainedwith cytosolic stains such as CMFDA produce a fluorescence intensitythat is proportional to cell thickness. Although cell labeling is dimmerin thin regions of the cell, total cell area can be discriminated frombackground. Fixed cells can be stained with cytoskeletal markers such asALEXA™ 488 phalloidin that label polymerized actin. Phalloidin does nothomogeneously stain the cytoplasm, but still permits discrimination ofthe total cell area from background.

[0194] Cellular hypertrophy

[0195] A screen to analyze cellular hypertrophy is implemented using thefollowing strategy. Primary rat myocytes can be cultured in 96 wellplates, treated with various compounds and then fixed and labeled with afluorescent marker for the cell membrane or cytoplasm, or cytoskeleton,such as an antibody to a cell surface marker or a fluorescent marker forthe cytoskeleton like rhodamine-phalloidin, in combination with a DNAlabel like Hoechst.

[0196] After focusing on the Hoechst labeled nuclei, two images areacquired, one of the Hoechst labeled nuclei and one of the fluorescentcytoplasm image. The nuclei are identified by thresholding to create amask and then comparing the morphological descriptors of the mask with aset of user defined descriptor values. Each non-nucleus image (or“cytoplasmic image”) is then processed separately. The originalcytoplasm image can be thresholded, creating a cytoplasmic mask image.Local regions containing cells are defined around the nuclei. The limitsof the cells in those regions are then defined by a local dynamicthreshold operation on the same region in the fluorescent antibodyimage. A sequence of erosions and dilations is used to separate slightlytouching cells and a second set of morphological descriptors is used toidentify single cells. The area of the individual cells is tabulated inorder to define the distribution of cell sizes for comparison with sizedata from normal and hypertrophic cells.

[0197] Responses from entire 96-well plates (measured as averagecytoplasmic area/cell) were analyzed by the above methods, and theresults demonstrated that the assay will perform the same on awell-to-well, plate-to-plate, and day-to-day basis (below a 15% cov formaximum signal). The data showed very good correlation for each day, andthat there was no variability due to well position in the plate.

[0198] The following totals can be computed for the field. The aggregatewhole nucleus area is the number of nonzero pixels in the nuclear mask.The average whole nucleus area is the aggregate whole nucleus areadivided by the total number of nuclei. For each cytoplasm image severalvalues can be computed. These are the total cytoplasmic area, which isthe count of nonzero pixels in the cytoplasmic mask. The aggregatecytoplasm intensity is the sum of the intensities of all pixels in thecytoplasmic mask. The cytoplasmic area per nucleus is the totalcytoplasmic area divided by the total nucleus count. The cytoplasmicintensity per nucleus is the aggregate cytoplasm intensity divided bythe total nucleus count. The average cytoplasm intensity is theaggregate cytoplasm intensity divided by the cytoplasm area. Thecytoplasm nucleus ratio is the total cytoplasm area divided by the totalnucleus area.

[0199] Additionally, one or more fluorescent antibodies to othercellular proteins, such as the major muscle proteins actin or myosin,can be included. Images of these additional labeled proteins can beacquired and stored with the above images, for later review, to identifyanomalies in the distribution and morphology of these proteins inhypertrophic cells. This example of a multi-parametric screen allows forsimultaneous analysis of cellular hypertrophy and changes in actin ormyosin distribution.

[0200] One of skill in the art will recognize that while the exampleanalyzes myocyte hypertrophy, the methods can be applied to analyzinghypertrophy, or general morphological changes in any cell type.

[0201] Cell Morphology Assays for Prostate Carcinoma

[0202] Cell spreading is a measure of the response of cell surfacereceptors to substrate attachment ligands. Spreading is proportional tothe ligand concentration or to the concentration of compounds thatreduce receptor-ligand function. One example of selective cell-substrateattachment is prostate carcinoma cell adhesion to the extracellularmatrix protein collagen. Prostate carcinoma cells metastasize to bonevia selective adhesion to collagen.

[0203] Compounds that interfere with metastasis of prostate carcinomacells were screened as follows. PC3 human prostate carcinoma cells werecultured in media with appropriate stimulants and are passaged tocollagen coated 96 well plates. Ligand concentration can be varied orinhibitors of cell spreading can be added to the wells. Examples ofcompounds that can affect spreading are receptor antagonists such asintegrin- or proteoglycan-blocking antibodies, signaling inhibitorsincluding phosphatidyl inositol-3 kinase inhibitors, and cytoskeletalinhibitors such as cytochalasin D. After two hours, cells were fixed andstained with ALEXA™ 488 phalloidin (Molecular Probes) and Hoechst 33342as per the protocol for cellular hypertrophy. The size of cells underthese various conditions, as measured by cytoplasmic staining, can bedistinguished above background levels. The number of cells per field isdetermined by measuring the number of nuclei stained with the HoechstDNA dye. The area per cell is found by dividing the cytoplasmic area(phalloidin image) by the cell number (Hoechst image). The size of cellsis proportional to the ligand-receptor function. Since the area isdetermined by ligand concentration and by the resultant function of thecell, drug efficacy, as well as drug potency, can be determined by thiscell-based assay. Other measurements can be made as discussed above forcellular hypertrophy.

[0204] The methods for analyzing cellular morphology can be used in acombined high throughput-high content screen. In one example, the highthroughput mode scans the whole well for an increase in fluorescentphalloidin intensity. A threshold is set above which both nuclei(Hoechst) and cells (phalloidin) are measured in a high content mode. Inanother example, an environmental biosensor (examples include, but arenot limited to, those biosensors that are sensitive to calcium and pHchanges) is added to the cells, and the cells are contacted with acompound. The cells are scanned in a high throughput mode, and thosewells that exceed a pre-determined threshold for luminescence of thebiosensor are scanned in a high content mode.

[0205] In a further aspect, kits are provided for analyzing cellularmorphology, comprising a luminescent compound that can be used tospecifically label the cell cytoplasm, membrane, or cytoskeleton (suchas those described above), and instructions for using the luminescentcompound to identify test stimuli that induce or inhibit changes incellular morphology according to the above methods. In a preferredembodiment, the kit further comprises a luminescent marker for cellnuclei. In a further preferred embodiment, the kit comprises at leastone compound that is known to modify cellular morphology, including, butnot limited to integrin- or proteoglycan-blocking antibodies, signalinginhibitors including phosphatidyl inositol-3 kinase inhibitors, andcytoskeletal inhibitors such as cytochalasin D.

[0206] In another aspect, the present invention comprises a machinereadable storage medium comprising a program containing a set ofinstructions for causing a cell screening system to execute thedisclosed methods for analyzing cellular morphology, wherein the cellscreening system comprises an optical system with a stage adapted forholding a plate containing cells, a digital camera, a means fordirecting fluorescence or luminescence emitted from the cells to thedigital camera, and a computer means for receiving and processing thedigital data from the digital camera.

EXAMPLE 3 Dual Mode High Throughput and High-Content Screen

[0207] The following example is a screen for activation of a G-proteincoupled receptor (GPCR) as detected by the translocation of the GPCRfrom the plasma membrane to a proximal nuclear location. This exampleillustrates how a high throughput screen can be coupled with ahigh-content screen in the dual mode System for Cell Based Screening.

[0208] G-protein coupled receptors are a large class of 7 trans-membranedomain cell surface receptors. Ligands for these receptors stimulate acascade of secondary signals in the cell, which may include, but are notlimited to, Ca⁺⁺ transients, cyclic AMP production, inositoltriphosphate (IP₃) production and phosphorylation. Each of these signalsare rapid, occuring in a matter of seconds to minutes, but are alsogeneric. For example, many different GPCRs produce a secondary Ca⁺⁺signal when activated. Stimulation of a GPCR also results in thetransport of that GPCR from the cell surface membrane to an internal,proximal nuclear compartment. This internalization is a much morereceptor-specific indicator of activation of a particular receptor thanare the secondary signals described above.

[0209]FIG. 19 illustrates a dual mode screen for activation of a GPCR.Cells carrying a stable chimera of the GPCR with a blue fluorescentprotein (BFP) would be loaded with the acetoxymethylester form ofFluo-3, a cell permeable calcium indicator (green fluorescence) that istrapped in living cells by the hydrolysis of the esters. They would thenbe deposited into the wells of a microtiter plate 601. The wells wouldthen be treated with an array of test compounds using a fluid deliverysystem, and a short sequence of Fluo-3 images of the whole microtiterplate would be acquired and analyzed for wells exhibiting a calciumresponse (i.e., high throughput mode). The images would appear like theillustration of the microtiter plate 601 in FIG. 19. A small number ofwells, such as wells C4 and E9 in the illustration, would fluoresce morebrightly due to the Ca⁺⁺ released upon stimulation of the receptors. Thelocations of wells containing compounds that induced a response 602would then be transferred to the HCS program and the optics switched fordetailed cell by cell analysis of the blue fluorescence for evidence ofGPCR translocation to the perinuclear region. The bottom of FIG. 19illustrates the two possible outcomes of the analysis of the highresolution cell data. The camera images a sub-region 604 of the wellarea 603, producing images of the fluorescent cells 605. In well C4, theuniform distribution of the fluorescence in the cells indicates that thereceptor has not internalized, implying that the Ca⁺⁺ response seen wasthe result of the stimulation of some other signalling system in thecell. The cells in well E9 606 on the other hand, clearly indicate aconcentration of the receptor in the perinuclear region clearlyindicating the full activation of the receptor. Because only a few hitwells have to be analyzed with high resolution, the overall throughputof the dual mode system can be quite high, comparable to the highthroughput system alone.

EXAMPLE 4 Kinetic High Content Screen

[0210] The following is an example of a screen to measure the kineticsof internalization of a receptor. As described above, the stimulation ofa GPCR, results in the internalization of the receptor, with a timecourse of about 15 min. Simply detecting the endpoint as internalized ornot, may not be sufficient for defining the potency of a compound as aGPCR agonist or antagonist. However, 3 time points at 5 min intervalswould provide information not only about potency during the time courseof measurement, but would also allow extrapolation of the data to muchlonger time periods. To perform this assay, the sub-region would bedefined as two rows, the sampling interval as 5 minutes and the totalnumber of time points 3. The system would then start by scanning tworows, and then adding reagent to the two rows, establishing the time=0reference. After reagent addition, the system would again scan the tworow sub-region acquiring the first time point data. Since this processwould take about 250 seconds, including scanning back to the beginningof the sub-region, the system would wait 50 seconds to begin acquisitionof the second time point. Two more cycles would produce the three timepoints and the system would move on to the second 2 row sub-region. Thefinal two 2-row sub-regions would be scanned to finish all the wells onthe plate, resulting in four time points for each well over the wholeplate. Although the time points for the wells would be offset slightlyrelative to time=0, the spacing of the time points would be very closeto the required 5 minutes, and the actual acquisition times and resultsrecorded with much greater precision than in a fixed-cell screen.

EXAMPLE 5 High-Content Screen of Human Glucocorticoid ReceptorTranslocation

[0211] One class of HCS involves the drug-induced dynamic redistributionof intracellular constituents. The human glucocorticoid receptor (hGR),a single “sensor” in the complex environmental response machinery of thecell, binds steroid molecules that have diffused into the cell. Theligand-receptor complex translocates to the nucleus wheretranscriptional activation occurs (Htun et al., Proc. Natl. Acad. Sci.93:4845, 1996).

[0212] In general, hormone receptors are excellent drug targets becausetheir activity lies at the apex of key intracellular signaling pathways.Therefore, a high-content screen of hGR translocation has distinctadvantage over in vitro ligand-receptor binding assays. The availabilityof up to two more channels of fluorescence in the cell screening systemof the present invention permits the screen to contain two additionalparameters in parallel, such as other receptors, other distinct targetsor other cellular processes.

[0213] Plasmid construct. A eukaryotic expression plasmid containing acoding sequence for a green fluorescent protein-human glucocorticoidreceptor (GFP-hGR) chimera was prepared using GFP mutants (Palm et al.,Nat. Struct. Biol. 4:361 (1997). The construct was used to transfect ahuman cervical carcinoma cell line (HeLa).

[0214] Cell preparation and transfection. HeLa cells (ATCC CCL-2) weretrypsinized and plated using DMEM containing 5% charcoal/dextran-treatedfetal bovine serum (FBS) (HyClone) and 1% penicillin-streptomycin(C-DMEM) 12-24 hours prior to transfection and incubated at 37° C. and5% CO₂. Transfections were performed by calcium phosphateco-precipitation (Graham and Van der Eb, Virology 52:456, 1973; Sambrooket al., (1989). Molecular Cloning: A Laboratory Manual, Second ed. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 1989) or withLipofectamine (Life Technologies, Gaithersburg, Md.). For the calciumphosphate transfections, the medium was replaced, prior to transfection,with DMEM containing 5% charcoal/dextran-treated FBS. Cells wereincubated with the calcium phosphate-DNA precipitate for 4-5 hours at37° C. and 5% CO₂, washed 3-4 times with DMEM to remove the precipitate,followed by the addition of C-DMEM.

[0215] Lipofectamine transfections were performed in serum-free DMEMwithout antibiotics according to the manufacturer's instructions (LifeTechnologies, Gaithersburg, Md.). Following a 2-3 hour incubation withthe DNA-liposome complexes, the medium was removed and replaced withC-DMEM. All transfected cells in 96-well microtiter plates wereincubated at 33° C. and 5% CO₂ for 24-48 hours prior to drug treatment.Experiments were performed with the receptor expressed transiently inHeLa cells.

[0216] Dexamethasone induction of GFP-hGR translocation. To obtainreceptor-ligand translocation kinetic data, nuclei of transfected cellswere first labeled with 5 μg/ml Hoechst 33342 (Molecular Probes) inC-DMEM for 20 minutes at 33° C. and 5% CO₂. Cells were washed once inHank's Balanced Salt Solution (HBSS) followed by the addition of 100 nMdexamethasone in HBSS with 1% charcoal/dextran-treated FBS. To obtainfixed time point dexamethasone titration data, transfected HeLa cellswere first washed with DMEM and then incubated at 33° C. and 5% CO₂ for1 h in the presence of 0-1000 nM dexamethasone in DMEM containing 1%charcoal/dextran-treated FBS. Cells were analyzed live or they wererinsed with HBSS, fixed for 15 min with 3.7% formaldehyde in HBSS,stained with Hoechst 33342, and washed before analysis. Theintracellular GFP-hGR fluorescence signal was not diminished by thisfixation procedure.

[0217] Image acquisition and analysis. Kinetic data were collected byacquiring fluorescence image pairs (GFP-hGR and Hoechst 33342-labelednuclei) from fields of living cells at 1 min intervals for 30 min afterthe addition of dexamethasone. Likewise, image pairs were obtained fromeach well of the fixed time point screening plates 1 h after theaddition of dexamethasone. In both cases, the image pairs obtained ateach time point were used to define nuclear and cytoplasmic regions ineach cell. Translocation of GFP-hGR was calculated by dividing theintegrated fluorescence intensity of GFP-hGR in the nucleus by theintegrated fluorescence intensity of the chimera in the cytoplasm or asa nuclear-cytoplasmic difference of GFP fluorescence. In the fixed timepoint screen this translocation ratio was calculated from data obtainedfrom at least 200 cells at each concentration of dexamethasone tested.Drug-induced translocation of GFP-hGR from the cytoplasm to the nucleuswas therefore correlated with an increase in the translocation ratio.

[0218] Results. FIG. 20 schematically displays the drug-inducedcytoplasm 253 to nucleus 252 translocation of the human glucocorticoidreceptor. The upper pair of schematic diagrams depicts the localizationof GFP-hGR within the cell before 250 (A) and after 251(B) stimulationwith dexamethasone. Under these experimental conditions, the druginduces a large portion of the cytoplasmic GFP-hGR to translocate intothe nucleus. This redistribution is quantified by determining theintegrated intensities ratio of the cytoplasmic and nuclear fluorescencein treated 255 and untreated 254 cells. The lower pair of fluorescencemicrographs show the dynamic redistribution of GFP-hGR in a single cell,before 254 and after 255 treatment. The HCS is performed on wellscontaining hundreds to thousands of transfected cells and thetranslocation is quantified for each cell in the field exhibiting GFPfluorescence. Although the use of a stably transfected cell line wouldyield the most consistently labeled cells, the heterogeneous levels ofGFP-hGR expression induced by transient transfection did not interferewith analysis by the cell screening system of the present invention.

[0219] To execute the screen, the cell screening system scans each wellof the plate, images a population of cells in each, and analyzes cellsindividually. Here, two channels of fluorescence are used to define thecytoplasmic and nuclear distribution of the GFP-hGR within each cell.

[0220] Depicted in FIG. 21 is the graphical user interface of the cellscreening system near the end of a GFP-hGR screen. The user interfacedepicts the parallel data collection and analysis capability of thesystem. The windows labeled “Nucleus” 261 and “GFP-hGR” 262 show thepair of fluorescence images being obtained and analyzed in a singlefield. The window labeled “Color Overlay” 260 is formed bypseudocoloring the above images and merging them so the user canimmediately identify cellular changes. Within the “Stored ObjectRegions” window 265, an image containing each analyzed cell and itsneighbors is presented as it is archived. Furthermore, as the HCS dataare being collected, they are analyzed, in this case for GFP-hGRtranslocation, and translated into an immediate “hit” response. The 96well plate depicted in the lower window of the screen 267 shows whichwells have met a set of user-defined screening criteria. For example, awhite-colored well 269 indicates that the drug-induced translocation hasexceeded a predetermined threshold value of 50%. On the other hand, ablack-colored well 270 indicates that the drug being tested induced lessthan 10% translocation. Gray-colored wells 268 indicate “hits” where thetranslocation value fell between 10% and 50%. Row “E” on the 96 wellplate being analyzed 266 shows a titration with a drug known to activateGFP-hGR translocation, dexamethasone. This example screen used only twofluorescence channels. Two additional channels (Channels 3 263 and 4264) are available for parallel analysis of other specific targets, cellprocesses, or cytotoxicity to create multiple parameter screens.

[0221] There is a link between the image database and the informationdatabase that is a powerful tool during the validation process of newscreens. At the completion of a screen, the user has total access toimage and calculated data (FIG. 22). The comprehensive data analysispackage of the cell screening system allows the user to examine HCS dataat multiple levels. Images 276 and detailed data in a spread sheet 279for individual cells can be viewed separately, or summary data can beplotted. For example, the calculated results of a single parameter for25- each cell in a 96 well plate are shown in the panel labeled Graph 1275. By selecting a single point in the graph, the user can display theentire data set for a particular cell that is recalled from an existingdatabase. Shown here are the image pair 276 and detailed fluorescenceand morphometric data from a single cell (Cell #118, gray line 277). Thelarge graphical insert 278 shows the results of dexamethasoneconcentration on the translocation of GFP-hGR. Each point is the averageof data from at least 200 cells. The calculated EC₅₀ for dexamethasonein this assay is 2 nM.

[0222] A powerful aspect of HCS with the cell screening system is thecapability of kinetic measurements using multicolor fluorescence andmorphometric parameters in living cells. Temporal and spatialmeasurements can be made on single cells within a population of cells ina field. FIG. 23 shows kinetic data for the dexamethasone-inducedtranslocation of GFP-hGR in several cells within a single field. HumanHeLa cells transfected with GFP-hGR were treated with 100 nMdexamethasone and the translocation of GFP-hGR was measured over time ina population of single cells. The graph shows the response oftransfected cells 285, 286, 287, and 288 and non-transfected cells 289.These data also illustrate the ability to analyze cells with differentexpression levels.

EXAMPLE 6 High-Content Screen of Drug-Induced Apoptosis

[0223] Apoptosis is a complex cellular program that involves myriadmolecular events and pathways. To understand the mechanisms of drugaction on this process, it is essential to measure as many of theseevents within cells as possible with temporal and spatial resolution.Therefore, an apoptosis screen that requires little cell samplepreparation yet provides an automated readout of severalapoptosis-related parameters would be ideal. A cell-based assay designedfor the cell screening system has been used to simultaneously quantifyseveral of the morphological, organellar, and macromolecular hallmarksof paclitaxel-induced apoptosis.

[0224] Cell preparation. The cells chosen for this study were mouseconnective tissue fibroblasts (L-929; ATCC CCL-1) and a highly invasiveglioblastoma cell line (SNB-19; ATCC CRL-2219) (Welch et al., In VitroCell. Dev. Biol. 31:610, 1995). The day before treatment with anapoptosis inducing drug, 3500 cells were placed into each well of a96-well plate and incubated overnight at 37° C. in a humidified 5% CO₂atmosphere. The following day, the culture medium was removed from eachwell and replaced with fresh medium containing various concentrations ofpaclitaxel (0-50 μM) from a 20 mM stock made in DMSO. The maximalconcentration of DMSO used in these experiments was 0.25%. The cellswere then incubated for 26 h as above. At the end of the paclitaxeltreatment period, each well received fresh medium containing 750 nMMitoTracker Red (Molecular Probes; Eugene, Oreg.) and 3 μg/ml Hoechst33342 DNA-binding dye (Molecular Probes) and was incubated as above for20 min. Each well on the plate was then washed with HBSS and fixed with3.7% formaldehyde in HBSS for 15 min at room temperature. Theformaldehyde was washed out with HBSS and the cells were permeabilizedfor 90 s with 0.5% (v/v) Triton X-100, washed with HBSS, incubated with2 U ml⁻¹ Bodipy FL phallacidin (Molecular Probes) for 30 min, and washedwith HBSS. The wells on the plate were then filled with 200 μl HBSS,sealed, and the plate stored at 4° C. if necessary. The fluorescencesignals from plates stored this way were stable for at least two weeksafter preparation. As in the nuclear translocation assay, fluorescencereagents can be designed to convert this assay into a live cellhigh-content screen.

[0225] Image acquisition and analysis on the ArrayScan System. Thefluorescence intensity of intracellular MitoTracker Red, Hoechst 33342,and Bodipy FL phallacidin was measured with the cell screening system asdescribed supra. Morphometric data from each pair of images obtainedfrom each well was also obtained to detect each object in the imagefield (e.g., cells and nuclei), and to calculate its size, shape, andintegrated intensity.

[0226] Calculations and output. A total of 50-250 cells were measuredper image field. For each field of cells, the following calculationswere performed: (1) The average nuclear area (μm²) was calculated bydividing the total nuclear area in a field by the number of nucleidetected. (2) The average nuclear perimeter (μm) was calculated bydividing the sum of the perimeters of all nuclei in a field by thenumber of nuclei detected in that field. Highly convoluted apoptoticnuclei had the largest nuclear perimeter values. (3) The average nuclearbrightness was calculated by dividing the integrated intensity of theentire field of nuclei by the number of nuclei in that field. Anincrease in nuclear brightness was correlated with increased DNAcontent. (4) The average cellular brightness was calculated by dividingthe integrated intensity of an entire field of cells stained withMitoTracker dye by the number of nuclei in that field. Because theamount of MitoTracker dye that accumulates within the mitochondria isproportional to the mitochondrial potential, an increase in the averagecell brightness is consistent with an increase in mitochondrialpotential. (5) The average cellular brightness was also calculated bydividing the integrated intensity of an entire field of cells stainedwith Bodipy FL phallacidin dye by the number of nuclei in that field.Because the phallotoxins bind with high affinity to the polymerized formof actin, the amount of Bodipy FL phallacidin dye that accumulateswithin the cell is proportional to actin polymerization state. Anincrease in the average cell brightness is consistent with an increasein actin polymerization.

[0227] Results. FIG. 24 (top panels) shows the changes paclitaxelinduced in the nuclear morphology of L-929 cells. Increasing amounts ofpaclitaxel caused nuclei to enlarge and fragment 293, a hallmark ofapoptosis. Quantitative analysis of these and other images obtained bythe cell screening system is presented in the same figure. Eachparameter measured showed that the L-929 cells 296 were less sensitiveto low concentrations of paclitaxel than were SNB-19 cells 297. Athigher concentrations though, the L-929 cells showed a response for eachparameter measured. The multiparameter approach of this assay is usefulin dissecting the mechanisms of drug action. For example, the area,brightness, and fragmentation of the nucleus 298 and actinpolymerization values 294 reached a maximum value when SNB-19 cells weretreated with 10 nM paclitaxel (FIG. 24; top and bottom graphs). However,mitochondrial potential 295 was minimal at the same concentration ofpaclitaxel (FIG. 24; middle graph). The fact that all the parametersmeasured approached control levels at increasing paclitaxelconcentrations (>10 nM) suggests that SNB-19 cells have low affinitydrug metabolic or clearance pathways that are compensatory atsufficiently high levels of the drug. Contrasting the drug sensitivityof SNB-19 cells 297, L-929 showed a different response to paclitaxel296. These fibroblastic cells showed a maximal response in manyparameters at 5 μM paclitaxel, a 500-fold higher dose than SNB-19 cells.Furthermore, the L-929 cells did not show a sharp decrease inmitochondrial potential 295 at any of the paclitaxel concentrationstested. This result is consistent with the presence of unique apoptosispathways between a normal and cancer cell line. Therefore, these resultsindicate that a relatively simple fluorescence labeling protocol can becoupled with the cell screening system of the present invention toproduce a high-content screen of key events involved in programmed celldeath.

BACKGROUND

[0228] A key to the mechanism of apoptosis was the discovery that,irrespective of the lethal stimulus, death results in identicalapoptotic morphology that includes cell and organelle dismantling andrepackaging, DNA cleavage to nucleosome sized fragments, and engulfinentof the fragmented cell to avoid an inflammatory response. Apoptosis istherefore distinct from necrosis, which is mediated more by acute traumato a cell, resulting in spillage of potentially toxic and antigeniccellular components into the intercellular milieu, leading to aninflammatory response.

[0229] The criteria for determining whether a cell is undergoingapoptosis (Wyllie et al. 1980. Int Rev Cytol. 68:251-306; Thompson,1995. Science. 267:1456-62; Majno and Joris. 1995. Am J Pathol.146:3-15; Allen et al. 1998. Cell Mol Life Sci. 54:427-45) includedistinct morphological changes in the appearance of the cell, as well asalterations in biochemical and molecular markers. For example, apoptoticcells often undergo cytoplasmic membrane blebbing, their chromosomesrapidly condense and aggregate around the nuclear periphery, the nucleusfragments, and small apoptotic bodies are formed. In many, but not all,apoptotic cells, chromatin becomes a target for specific nucleases thatcleave the DNA.

[0230] Apoptosis is commonly accompanied by a characteristic change innuclear morphology (chromatin condensation or fragmentation) and astep-wise fragmentation of DNA culminating in the formation of mono-and/or oligomeric fragments of 200 base pairs. Specific changes inorganellar function, such as mitochondrial membrane potential, occur. Inaddition, specific cysteine proteases (caspases) are activated, whichcatalyzes a highly selective pattern of protein degradation byproteolytic cleavage after specific aspartic acid residues. In addition,the external surface exposure of phosphatidylserine residues (normallyon the inner membrane leaflet) allows for the recognition andelimination of apoptotic cells, before the membrane breaks up andcytosol or organelles spill into the intercellular space and elicitinflammatory reactions. Moreover, cells undergoing apoptosis tend toshrink, while also having a reduced intracellular potassium level.

[0231] The general patterns of apoptotic signals are very similar amongdifferent cell types and apoptotic inducers. However, the details of thepathways actually vary significantly depending on cell type and inducer.The dependence and independence of various signal transduction pathwaysinvolved in apoptosis are currently topics of intense research. We showhere that the pathway also varies depending upon the dose of the inducerin specific cell types.

[0232] Nuclear Morphology

[0233] Cells undergoing apoptosis generally exhibit two types of nuclearchange, fragmentation or condensation ((Majno and Joris, 1995),(Earnshaw, 1995)). The response in a given cell type appears to varydepending on the apoptotic inducer. During nuclear fragmentation, acircular or oval nucleus becomes increasingly lobular. Eventually, thenucleus fragments dramatically into multiple sub-nuclei. Sometimes thedensity of the chromatin within the lobular nucleus may show spatialvariations in distribution (heterochromatization), approximating themargination seen in nuclear condensation.

[0234] Nuclear condensation has been reported in some cell types, suchas MCF-7 (Saunders et al. 1997. Int J Cancer. 70:214-20). Condensationappears to arise as a consequence of the loss of structural integrity ofthe euchromatin, nuclear matrix and nuclear lamina (Hendzel et al. 1998.J. Biol. Chem. 273:24470-8). During nuclear condensation, the chromatinconcentrates near the margin of the nucleus, leading to the overallshrinkage of the nucleus. Thus, the use of nuclear morphology as ameasure of apoptosis must take both condensation and fragmentation intoaccount.

[0235] Material and Methods

[0236] Cells were plated into 96-well plates at densities of 3×10³ to1×10⁴ cells/well. The following day apoptotic inducers were added atindicated concentrations and cells were incubated for indicated timeperiods (usually 16-30 hours). The next day medium was removed and cellswere stained with 5 μg/ml Hoechst (Molecular Probes, Inc.) in freshmedium and incubated for 30 minutes at 37° C. Cells were washed inHank's Balanced Salt Solution (HBSS) and fixed with 3.7% formaldehyde inHBSS at room temperature. Cells were washed 2×with HBSS at roomtemperature and the plate was sealed.

[0237] Quantitation of changes in nuclear morphology upon induction ofapoptosis was accomplished by (1) measuring the effective size of thenuclear region; and (2) measuring the degree of convolution of theperimeter. The size parameter provides the more sensitive measure ofnuclear condensation, whereas the perimeter measure provides a moresensitive measure of nuclear fragmentation.

[0238] Results & Discussion

[0239] L929 cells responded to both staurosporine (30 hours) andpaclitaxel (30 hours) with a dose-dependent change in nuclear morphology(FIGS. 25A and 25B). BHK cells illustrated a slightly more complicated,yet clearly visible response. Staurosporine appeared to stimulatenuclear condensation at lower doses and nuclear fragmentation at higherdoses (FIGS. 25C and 25D). In contrast, paclitaxel induced a consistentincrease in nuclear fragmentation with increasing concentrations. Theresponse of MCF-7 cells varied dramatically depending upon the apoptoticinducer. Staurosporine appeared to elicit nuclear condensation whereaspaclitaxel induced nuclear fragmentation (FIGS. 25E and 25F).

[0240]FIG. 26 illustrates the dose response of cells in terms of bothnuclear size and nuclear perimeter convolution. There appears to be aswelling of the nuclei that precedes the fragmentation.

[0241] Result of evaluation: Differential responses by cell lines and byapoptotic inducers were observed in a dose dependent manner, indicatingthat this assay will be useful for detecting changes in the nucleuscharacteristic of apoptosis.

[0242] Actin Reorganization

[0243] We assessed changes in the actin cytoskeleton as a potentialparameter related to apoptotic changes. This was based on preliminaryobservations of an early increase in f-actin content detected withfluorescent phalloidin labeling, an f-actin specific stain (ourunpublished data; Levee et al. 1996. Am J Physiol. 271:C1981-92; Maekawaet al. 1996. Clin Exp Immunol. 105:389-96). Changes in the actincytoskeleton during apoptosis have not been observed in all cell types.(Endresen et al. 1995. Cytometry. 20:162-71, van Engeland et al. 1997.Exp Cell Res. 235:421-30).

[0244] Material and Methods

[0245] Cells were plated in 96-well plates at densities of 3×10³ to1×10⁴ cells/well. The following day apoptotic inducers were added atindicated concentrations. Cells were incubated for the indicated timeperiods (usually 16-30 hours). The next day the medium was removed andcells were stained with 5 μg/ml Hoechst (Molecular Probes, Inc.) infresh medium and incubated for 30 minutes at 30° C. Cells were washed inHBSS and fixed with 3.7% formaldehyde in HBSS at room temperature.Plates were washed with HBSS and permeabilized with 0.5% v/v TritonX-100 in HBSS at room temperature. Plates were washed in HBSS andstained with 100 μl of 1 U/ml of Alexa 488 Phalloidin stock (100μl/well, Molecular Probes, Inc.). Cells were washed 2×with HBSS at RTand the plate was sealed.

[0246] Quantitation of f-actin content was accomplished by measuring theintensity of phalloidin staining around the nucleus. This was determinedto be a reasonable approximation of a full cytoplasmic average of theintensity. The mask used to approximate this cytoplasmic measure wasderived from the nuclear mask defined by the Hoechst stain. Derivationwas accomplished by combinations of erosions and dilations.

[0247] Results and Discussion

[0248] Changes in f-actin content varied based on cell type andapoptotic inducer (FIG. 27). Staurosporine (30 hours) induced increasesin f-actin in L929 (FIG. 27A) and BHK (FIG. 27B) cells. MCF-7 cellsexhibited a concentration-dependent response. At low concentrations(FIG. 27E) there appeared to be a decrease in f-actin content. At higherconcentrations, f-actin content increased. Paclitaxel (30 hours)treatment led to a wide variety of responses. L929 cells responded withgraded increases in f-actin (FIG. 27B) whereas both BHK and MCF-7responses were highly variable (FIGS. 27D & 27F, respectively).

[0249] Result of Evaluation: Both increases and decreases in signalintensity were measured for several cell lines and found to exhibit aconcentration dependent response. For certain cell line/apoptoticinducer pairs this could be a statistically significant apoptoticindicator.

[0250] Changes in Mitochondrial Mass/Potential

[0251] Introduction

[0252] Changes in mitochondria play a central role in apoptosis (Henkartand Grinstein. 1996. J Exp Med. 183:1293-5). Mitochondria releaseapoptogenic factors through the outer membrane and dissipate theelectrochemical gradient of the inner membrane. This is thought to occurvia formation of the mitochondria permeability transition (MPT),although it is apparently not true in all cases. An obviousmanifestation of the formation of the MPT is collapse of themitochondrial membrane potential. Inhibition of MPT by pharmacologicalintervention or mitochondrial expression of the anti-apoptotic proteinBcl-2 prevents cell death, suggesting the formation of the MPT may be arate-limiting event of the death process (For review see: Kroemer et al.1998. Annu Rev Physiol. 60:619-42). It has also been observed thatmitochondria can proliferate during stimulation of apoptosis (Mancini etal. 1997. J. Cell Biol. 138:449-69; Camilleri-Broet et al. 1998. ExpCell Res. 239:277-92).

[0253] One approach for measuring apoptosis-induced changes inmitochondria is to measure the mitochondrial membrane potential. Of themethods available, the simplest measure is the redistribution of acationic dye that distributes within intracellular organelles based onthe membrane potential. Such an approach traditionally requires livecells for the measurements. The recent introduction of the MitoTrackerdyes (Poot et al. 1997. Cytometry. 27:358-64; available from MolecularProbes, Inc., Oregon) provides a means of measuring mitochondrialmembrane potential after fixation.

[0254] Given the observations of a possible increase in mitochondrialmass during apoptosis, the amount of dye labeling the mitochondria isrelated to both membrane potential and the number of mitochondria. Ifthe number of mitochondria remains constant then the amount of dye isdirectly related to the membrane potential. If the number ofmitochondria is not constant, then the signal will likely be dominatedby the increase in mass (Reipert et al. 1995. Exp Cell Res. 221:281-8).

[0255] Probes are available that allow a clear separation betweenchanges in mass and potential in HCS assays. Mitochondrial mass ismeasured directly by labeling with Mitotracker Green FM (Poot andPierce, 1999, Cytometry. 35:311-7; available from Molecular Probes,Inc., Oregon). The labeling is independent of mitochondrial membranepotential but proportional to mitochondrial mass. This also provides ameans of normalizing other mitochondrial measures in each cell withrespect to mitochondrial mass.

[0256] Material and Methods

[0257] Cells were plated into 96-well plates at densities of 3×10³ to1×10⁴ cells/well. The following day apoptotic inducers were added at theindicated concentrations and cells were incubated for the indicated timeperiods (usually 16-30 hours). Cells were stained with 5 μg/ml Hoechst(Molecular Probes, Inc.) and 750 nM MitoTracker Red (CMXRos, MolecularProbes, Inc.) in fresh medium and incubated for 30 minutes at 37° C.Cells were washed in HBSS and fixed with 3.7% formaldehyde in HBSS atroom temperature. Plates were washed with HBSS and permeabilized with0.5% v/v Triton X-100 in HBSS at room temperature. Cells were washed2×with HBSS at room temperature and the plate was sealed. For duallabeling of mitochondria, cells were treated with 200 nM MitotrackerGreen and 200 nM Mitotracker Red for 0.5 hours before fixation.

[0258] Results & Discussion

[0259] Induction of apoptosis by staurosporine and paclitaxel led tovarying mitochondrial changes depending upon the stimulus. L929 cellsexhibited a clear increase in mitochondrial mass with increasingstaurosporine concentrations (FIG. 28). BHK cells exhibited either adecrease in membrane potential at lower concentrations of staurosporine,or an increase in mass at higher concentrations of staurosporine (FIG.28C). MCF-7 cells responded by a consistent decrease in mitochondrialmembrane potential in response to increasing concentrations ofstaurosporine (FIG. 28E). Increasing concentrations of paclitaxel causedconsistent increases in mitochondrial mass (FIGS. 28B, 28D, and 28F).

[0260] The mitochondrial membrane potential is measured by labelingmitochondria with both Mitotracker Green FM and Mitotracker Red(Molecular Probes, Inc). Mitotracker Red labeling is proportional toboth mass and membrane potential. Mitotracker Green FM labeling isproportional to mass. The ratio of Mitotracker Red signal to theMitotracker Green FM signal provides a measure of mitochondrial membranepotential (Poot and Pierce, 1999). This ratio normalizes themitochondrial mass with respect to the Mitotracker Red signal. (See FIG.28G) Combining the ability to normalize to mitochondrial mass with ameasure of the membrane potential allows independent assessment of bothparameters.

[0261] Result of Evaluation: Both decreases in potential and increasesin mass were observed depending on the cell line and inducer tested.Dose dependent correlation demonstrates that this is a promisingapoptotic indicator.

[0262] It is possible to combine multiple measures of apoptosis byexploiting the spectral domain of fluorescence spectroscopy. In fact,all of the nuclear morphology/f-actin content/mitochondrialmass/mitochondrial potential data shown earlier were collected asmultiparameter assays, but were presented individually for clarity.

EXAMPLE 7 Protease Induced Translocation of a Signaling EnzymeContaining a Disease-Associated Sequence From Cytoplasm to Nucleus

[0263] Plasmid construct. A eukaryotic expression plasmid containing acoding sequence for a green fluorescent protein—caspase (Cohen (1997),Biochemical J. 326:1-16; Liang et al. (1997), J. of Molec. Biol.274:291-302) chimera is prepared using GFP mutants. The construct isused to transfect eukaryotic cells.

[0264] Cell preparation and transfection. Cells are trypsinized andplated 24 h prior to transfection and incubated at 37° C. and 5% CO₂.Transfections are performed by methods including, but not limited tocalcium phosphate coprecipitation or lipofection. Cells are incubatedwith the calcium phosphate-DNA precipitate for 4-5 hours at 37° C. and5% CO₂, washed 3-4 times with DMEM to remove the precipitate, followedby the addition of C-DMEM. Lipofectamine transfections are performed inserum-free DMEM without antibiotics according to the manufacturer'sinstructions. Following a 2-3 hour incubation with the DNA-liposomecomplexes, the medium is removed and replaced with C-DMEM.

[0265] Apopototic induction of Caspase-GFP translocation. To obtainCaspase-GFP translocation kinetic data, nuclei of transfected cells arefirst labeled with 5 μg/ml Hoechst 33342 (Molecular Probes) in C-DMEMfor 20 minutes at 37° C. and 5% CO₂. Cells are washed once in Hank'sBalanced Salt Solution (HBSS) followed by the addition of compounds thatinduce apoptosis. These compounds include, but are not limited topaclitaxel, staurosporine, ceramide, and tumor necrosis factor. Toobtain fixed time point titration data, transfected cells are firstwashed with DMEM and then incubated at 37° C. and 5% CO₂ for 1 h in thepresence of 0-1000 nM compound in DMEM. Cells are analyzed live or theyare rinsed with HBSS, fixed for 15 min with 3.7% formaldehyde in HBSS,stained with Hoechst 33342, and washed before analysis.

[0266] Image acquisition and analysis. Kinetic data are collected byacquiring fluorescence image pairs (Caspase-GFP and Hoechst33342-labeled nuclei) from fields of living cells at 1 min intervals for30 min after the addition of compound. Likewise, image pairs areobtained from each well of the fixed time point screening plates 1 hafter the addition of compound. In both cases, the image pairs obtainedat each time point are used to define nuclear and cytoplasmic regions ineach cell. Translocation of Caspase-GFP is calculated by dividing theintegrated fluorescence intensity of Caspase-GFP in the nucleus by theintegrated fluorescence intensity of the chimera in the cytoplasm or asa nuclear-cytoplasmic difference of GFP fluorescence. In the fixed timepoint screen this translocation ratio is calculated from data obtainedfrom at least 200 cells at each concentration of compound tested.Drug-induced translocation of Caspase-GFP from the cytoplasm to thenucleus is therefore correlated with an increase in the translocationratio. Molecular interaction libraries including, but not limited tothose comprising putative activators or inhibitors ofapoptosis-activated enzymes are use to screen the indicator cell linesand identify a specific ligand for the DAS, and a pathway activated bycompound activity.

EXAMPLE 8 Identification of Novel Steroid Receptors From DAS

[0267] Two sources of material and/or information are required to makeuse of this embodiment, which allows assessment of the function of anuncharacterized gene. First, disease associated sequence bank(s)containing cDNA sequences suitable for transfection into mammalian cellscan be used. Because every RADE or differential expression experimentgenerates up to several hundred sequences, it is possible to generate anample supply of DAS. Second, information from primary sequence databasesearches can be used to place DAS into broad categories, including, butnot limited to, those that contain signal sequences, seventrans-membrane motifs, conserved protease active site domains, or otheridentifiable motifs. Based on the information acquired from thesesources, method types and indicator cell lines to be transfected areselected. A large number of motifs are already well characterized andencoded in the linear sequences contained within the large number genesin existing genomic databases.

[0268] In one embodiment, the following steps are taken:

[0269] 1) Information from the DAS identification experiment (includingdatabase searches) is used as the basis for selecting the relevantbiological processes. (for example, look at the DAS from a tumor linefor cell cycle modulation, apoptosis, metastatic proteases, etc.)

[0270] 2) Sorting of DNA sequences or DAS by identifiable motifs (ie.signal sequences, 7-transmembrane domains, conserved protease activesite domains, etc.) This initial grouping will determine fluorescenttagging strategies, host cell lines, indicator cell lines, and banks ofbioactive molecules to be screened, as described supra.

[0271] 3) Using well established molecular biology methods, ligate DASinto an expression vector designed for this purpose. Generalizedexpression vectors contain promoters, enhancers, and terminators forwhich to deliver target sequences to the cell for transient expression.Such vectors may also contain antibody tagging sequences, directassociation sequences, chromophore fusion sequences like GFP, etc. tofacilitate detection when expressed by the host.

[0272] 4) Transiently transfect cells with DAS containing vectors usingstandard transfection protocols including: calcium phosphateco-precipitation, liposome mediated, DEAE dextran mediated, polycationicmediated, viral mediated, or electroporation, and plate into microtiterplates or microwell arrays. Alternatively, transfection can be donedirectly in the microtiter plate itself.

[0273] 5) Carry out the cell screening methods as described supra.

[0274] In this embodiment, DAS shown to possess a motif(s) suggestive oftranscriptional activation potential (for example, DNA binding domain,amino terminal modulating domain, hinge region, or carboxy terminalligand binding domain) are utilized to identify novel steroid receptors.

[0275] Defining the fluorescent tags for this experiment involvesidentification of the nucleus through staining, and tagging the DAS bycreating a GFP chimera via insertion of DAS into an expression vector,proximally fused to the gene encoding GFP. Alternatively, a single chainantibody fragment with high affinity to some portion of the expressedDAS could be constructed using technology available in the art(Cambridge Antibody Technologies) and linked to a fluorophore (FITC) totag the putative transcriptional activator/receptor in the cells. Thisalternative would provide an external tag requiring no DNA transfectionand therefore would be useful if distribution data were to be gatheredfrom the original primary cultures used to generate the DAS.

[0276] Plasmid construct. A eukaryotic expression plasmid containing acoding sequence for a green fluorescent protein—DAS chimera is preparedusing GFP mutants. The construct is used to transfect HeLa cells. Theplasmid, when transfected into the host cell, produces a GFP fused tothe DAS protein product, designated GFP-DASpp.

[0277] Cell preparation and transfection. HeLa cells are trypsinized andplated using DMEM containing 5% charcoal/dextran-treated fetal bovineserum (FBS) (Hyclone) and 1% penicillin-streptomycin (C-DMEM) 12-24hours prior to transfection and incubated at 37° C. and 5% CO₂.Transfections are performed by calcium phosphate coprecipitation or withLipofectamine (Life Technologies). For the calcium phosphatetransfections, the medium is replaced, prior to transfection, with DMEMcontaining 5% charcoal/dextran-treated FBS. Cells are incubated with thecalcium phosphate-DNA precipitate for 4-5 hours at 37° C. and 5% CO₂,and washed 3-4 times with DMEM to remove the precipitate, followed bythe addition of C-DMEM. Lipofectamine transfections are performed inserum-free DMEM without antibiotics according to the manufacturer'sinstructions. Following a 2-3 hour incubation with the DNA-liposomecomplexes, the medium is removed and replaced with C-DMEM. Alltransfected cells in 96-well microtiter plates are incubated at 33° C.and 5% CO₂ for 24-48 hours prior to drug treatment. Experiments areperformed with the receptor expressed transiently in HeLa cells.

[0278] Localization of expressed GFP-DASpp inside cells. To obtaincellular distribution data, nuclei of transfected cells are firstlabeled with 5 μg/ml Hoechst 33342 (Molecular Probes) in C-DMEM for 20minutes at 33° C. and 5% CO₂. Cells are washed once in Hank's BalancedSalt Solution (HBSS). The cells are analyzed live or they are rinsedwith HBSS, fixed for 15 min with 3.7% formaldehyde in HBSS, stained withHoechst 33342, and washed before analysis.

[0279] In a preferred embodiment, image acquisition and analysis areperformed using the cell screening system of the present invention. Theintracellular GFP-DASpp fluorescence signal is collected by acquiringfluorescence image pairs (GFP-DASpp and Hoechst 33342-labeled nuclei)from field cells. The image pairs obtained at each time point are usedto define nuclear and cytoplasmic regions in each cell. Datademonstrating dispersed signal in the cytoplasm would be consistent withknown steroid receptors that are DNA transcriptional activators.

[0280] Screening for induction of GFP-DASpp translocation. Using theabove construct, confirmed for appropriate expression of the GFP-DASpp,as an indicator cell line, a screen of various ligands is performedusing a series of steroid type ligands including, but not limited to:estrogen, progesterone, retinoids, growth factors, androgens, and manyother steroid and steroid based molecules. Image acquisition andanalysis are performed using the cell screening system of the invention.The intracellular GFP-DASpp fluorescence signal is collected byacquiring fluorescence image pairs (GFP-DASpp and Hoechst 33342-labelednuclei) from fields cells. The image pairs obtained at each time pointare used to define nuclear and cytoplasmic regions in each cell.Translocation of GFP-DASpp is calculated by dividing the integratedfluorescence intensity of GFP-DASpp in the nucleus by the integratedfluorescence intensity of the chimera in the cytoplasm or as anuclear-cytoplasmic difference of GFP fluorescence. A translocation fromthe cytoplasm into the nucleus indicates a ligand binding activation ofthe DASpp thus identifying the potential receptor class and action.Combining this data with other data obtained in a similar fashion usingknown inhibitors and modifiers of steroid receptors, would eithervalidate the DASpp as a target, or more data would be generated fromvarious sources.

EXAMPLE 9 Additional Screens

[0281] Translocation Between the Plasma Membrane and the Cytoplasm:

[0282] Profilactin complex dissociation and binding of profilin to theplasma membrane. In one embodiment, a fluorescent protein biosensor ofprofilin membrane binding is prepared by labeling purified profilin(Federov et al.(1994), J. Molec. Biol. 241:480-482; Lanbrechts et al.(1995), Eur. J. Biochem. 230:281-286) with a probe possessing afluorescence lifetime in the range of 2-300 ns. The labeled profilin isintroduced into living indicator cells using bulk loading methodologyand the indicator cells are treated with test compounds. Fluorescenceanisotropy imaging microscopy (Gough and Taylor (1993), J. Cell Biol.121:1095-1107) is used to measure test-compound dependent movement ofthe fluorescent derivative of profilin between the cytoplasm andmembrane for a period of time after treatment ranging from 0.1 s to 10h.

[0283] Rho-RhoGDI complex translocation to the membrane. In anotherembodiment, indicator cells are treated with test compounds and thenfixed, washed, and permeabilized. The indicator cell plasma membrane,cytoplasm, and nucleus are all labeled with distinctly colored markersfollowed by immunolocalization of Rho protein (Self et al. (1995),Methods in Enzymology 256:3-10; Tanaka et al. (1995), Methods inEnzymology 256:41-49) with antibodies labeled with a fourth color. Eachof the four labels is imaged separately using the cell screening system,and the images used to calculate the amount of inhibition or activationof translocation effected by the test compound. To do this calculation,the images of the probes used to mark the plasma membrane and cytoplasmare used to mask the image of the immunological probe marking thelocation of intracellular Rho protein. The integrated brightness perunit area under each mask is used to form a translocation quotient bydividing the plasma membrane integrated brightness/area by thecytoplasmic integrated brightness/area. By comparing the translocationquotient values from control and experimental wells, the percenttranslocation is calculated for each potential lead compound.

[0284] β-Arrestin Translocation to the Plasma Membrane Upon G-ProteinReceptor Activation.

[0285] In another embodiment of a cytoplasm to membrane translocationhigh-content screen, the translocation of β-arrestin protein from thecytoplasm to the plasma membrane is measured in response to celltreatment. To measure the translocation, living indicator cellscontaining luminescent domain markers are treated with test compoundsand the movement of the β-arrestin marker is measured in time and spaceusing the cell screening system of the present invention. In a preferredembodiment, the indicator cells contain luminescent markers consistingof a green fluorescent protein β-arrestin (GFP-β-arrestin) proteinchimera (Barak et al. (1997), J. Biol. Chem. 272:27497-27500; Daaka etal. (1998), J. Biol. Chem. 273:685-688) that is expressed by theindicator cells through the use of transient or stable cell transfectionand other reporters used to mark cytoplasmic and membrane domains. Whenthe indicator cells are in the resting state, the domain markermolecules partition predominately in the plasma membrane or in thecytoplasm. In the high-content screen, these markers are used todelineate the cell cytoplasm and plasma membrane in distinct channels offluorescence. When the indicator cells are treated with a test compound,the dynamic redistribution of the GFP-β-arrestin is recorded as a seriesof images over a time scale ranging from 0.1 s to 10 h. In a preferredembodiment, the time scale is 1 h. Each image is analyzed by a methodthat quantifies the movement of the GFP-β-arrestin protein chimerabetween the plasma membrane and the cytoplasm. To do this calculation,the images of the probes used to mark the plasma membrane and cytoplasmare used to mask the image of the GFP-β-arrestin probe marking thelocation of intracellular GFP-β-arrestin protein. The integratedbrightness per unit area under each mask is used to form a translocationquotient by dividing the plasma membrane integrated brightness/area bythe cytoplasmic integrated brightness/area. By comparing thetranslocation quotient values from control and experimental wells, thepercent translocation is calculated for each potential lead compound.The output of the high-content screen relates quantitative datadescribing the magnitude of the translocation within a large number ofindividual cells that have been treated with test compounds of interest.

[0286] Translocation Between the Endoplasmic Reticulum and the Golgi:

[0287] In one embodiment of an endoplasmic reticulum to Golgitranslocation high-content screen, the translocation of a VSVG proteinfrom the ts045 mutant strain of vesicular stomatitis virus (Ellenberg etal. (1997), J. Cell Biol. 138:1193-1206; Presley et al. (1997) Nature389:81-85) from the endoplasmic reticulum to the Golgi domain ismeasured in response to cell treatment. To measure the translocation,indicator cells containing luminescent reporters are treated with testcompounds and the movement of the reporters is measured in space andtime using the cell screening system of the present invention. Theindicator cells contain luminescent reporters consisting of a GFP-VSVGprotein chimera that is expressed by the indicator cell through the useof transient or stable cell transfection and other domain markers usedto measure the localization of the endoplasmic reticulum and Golgidomains. When the indicator cells are in their resting state at 40° C.,the GFP-VSVG protein chimera molecules are partitioned predominately inthe endoplasmic reticulum. In this high-content screen, domain markersof distinct colors used to delineate the endoplasmic reticulum and theGolgi domains in distinct channels of fluorescence. When the indicatorcells are treated with a test compound and the temperature issimultaneously lowered to 32° C., the dynamic redistribution of theGFP-VSVG protein chimera is recorded as a series of images over a timescale ranging from 0.1 s to 10 h. Each image is analyzed by a methodthat quantifies the movement of the GFP-VSVG protein chimera between theendoplasmic reticulum and the Golgi domains. To do this calculation, theimages of the probes used to mark the endoplasmic reticulum and theGolgi domains are used to mask the image of the GFP-VSVG probe markingthe location of intracellular GFP-VSVG protein. The integratedbrightness per unit area under each mask is used to form a translocationquotient by dividing the endoplasmic reticulum integratedbrightness/area by the Golgi integrated brightness/area. By comparingthe translocation quotient values from control and experimental wells,the percent translocation is calculated for each potential leadcompound. The output of the high-content screen relates quantitativedata describing the magnitude of the translocation within a large numberof individual cells that have been treated with test compounds ofinterest at final concentrations ranging from 10⁻¹² M to 10⁻³ M for aperiod ranging from 1 min to 10 h.

[0288] Induction and Inhibition of Organellar Function:

[0289] Intracellular Microtubule Stability.

[0290] In another aspect of the invention, an automated method foridentifying compounds that modify microtubule structure is provided. Inthis embodiment, indicator cells are treated with test compounds and thedistribution of luminescent microtubule-labeling molecules is measuredin space and time using a cell screening system, such as the onedisclosed above. The luminescent microtubule-labeling molecules may beexpressed by or added to the cells either before, together with, orafter contacting the cells with a test compound.

[0291] In one embodiment of this aspect of the invention, living cellsexpress a luminescently labeled protein biosensor of microtubuledynamics, comprising a protein that labels microtubules fused to aluminescent protein. Appropriate microtubule-labeling proteins for thisaspect of the invention include, but are not limited to α and β tubulinisoforms, and MAP4. Preferred embodiments of the luminescent proteininclude, but are not limited to green fluorescent protein (GFP) and GFPmutants. In a preferred embodiment, the method involves transfectingcells with a microtubule labeling luminescent protein, wherein themicrotubule labeling protein can be, but is not limited to, α-tubulin,β-tubulin, or microtubule-associated protein 4 (MAP4). The approachoutlined here enables those skilled in the art to make live cellmeasurements to determine the effect of lead compounds on tubulinactivity and microtubule stability in vivo.

[0292] In a most preferred embodiment, MAP4 is fused to a modifiedversion of the Aequorea victoria green fluorescent protein (GFP). A DNAconstruct has been made which consists of a fusion between the EGFPcoding sequence (available from Clontech) and the coding sequence formouse MAP4. (Olson et al., (1995), J. Cell Biol. 130(3): 639-650). MAP4is a ubiquitous microtubule-associated protein that is known to interactwith microtubules in interphase as well as mitotic cells (Olmsted andMurofushi, (1993), MAP4. In “Guidebook to the Cytoskeleton and MotorProteins.” Oxford University Press. T. Kreis and R. Vale, eds.) Itslocalization, then, can serve as an indicator of the localization,organization, and integrity of microtubules in living (or fixed) cellsat all stages of the cell cycle for cell-based HCS assays. While MAP2and tau (microtubule associated proteins expressed specifically inneuronal cells) have been used to form GFP chimeras (Kaech et al.,(1996) Neuron. 17: 1189-1199; Hall et al., (1997), Proc. Nat. Acad. Sci.94: 4733-4738) their restricted cell type distribution and the tendencyof these proteins to bundle microtubules when overexpressed make theseproteins less desirable as molecular reagents for analysis in live cellsoriginating from varied tissues and organs. Moderate overexpression ofGFP-MAP4 does not disrupt microtubule function or integrity (Olson etal., 1995). Similar constructs can be made using p-tubulin or β-tubulinvia standard techniques in the art. These chimeras will provide a meansto observe and analyze microtubule activity in living cells during allstages of the cell cycle.

[0293] In another embodiment, the luminescently labeled proteinbiosensor of microtubule dynamics is expressed, isolated, and added tothe cells to be analyzed via bulk loading techniques, such asmicroinjection, scrape loading, and impact-mediated loading. In thisembodiment, there is not an issue of overexpression within the cell, andthus α and β tubulin isoforms, MAP4, MAP2 and/or tau can all be used.

[0294] In a further embodiment, the protein biosensor is expressed bythe cell, and the cell is subsequently contacted with a luminescentlabel, such as a labeled antibody, that detects the protein biosensor,endogenous levels of a protein antigen, or both. In this embodiment, aluminescent label that detects α and β tubulin isoforms, MAP4, MAP2and/or tau, can be used.

[0295] A variety of GFP mutants are available, all of which would beeffective in this invention, including, but not limited to, GFP mutantswhich are commercially available (Clontech, Calif.).

[0296] The MAP4 construct has been introduced into several mammaliancell lines (BHK-21, Swiss 3T3, HeLa, HEK 293, LLCPK) and theorganization and localization of tubulin has been visualized in livecells by virtue of the GFP fluorescence as an indicator of MAP4localization. The construct can be expressed transiently or stable celllines can be prepared by standard methods. Stable HeLa cell linesexpressing the EGFP-MAP4 chimera have been obtained, indicating thatexpression of the chimera is not toxic and does not interfere withmitosis.

[0297] Possible selectable markers for establishment and maintenance ofstable cell lines include, but are not limited to the neomycinresistance gene, hygromycin resistance gene, zeocin resistance gene,puromycin resistance gene, bleomycin resistance gene, and blastacidinresistance gene.

[0298] The utility of this method for the monitoring of microtubuleassembly, disassembly, and rearrangement has been demonstrated bytreatment of transiently and stably transfected cells with microtubuledrugs such as paclitaxel, nocodazole, vincristine, or vinblastine.

[0299] The present method provides high-content and combined highthroughput-high content cell-based screens for anti-microtubule drugs,particularly as one parameter in a multi-parametric cancer targetscreen. The EGFP-MAP4 construct used herein can also be used as one ofthe components of a high-content screen that measures multiple signalingpathways or physiological events. In a preferred embodiment, a combinedhigh throughput and high content screen is employed, wherein multiplecells in each of the locations containing cells are analyzed in a highthroughput mode, and only a subset of the locations containing cells areanalyzed in a high content mode. The high throughput screen can be anyscreen that would be useful to identify those locations containing cellsthat should be further analyzed, including, but not limited to,identifying locations with increased luminescence intensity, thoseexhibiting expression of a reporter gene, those undergoing calciumchanges, and those undergoing pH changes.

[0300] In addition to drug screening applications, the present inventionmay be applied to clinical diagnostics, the detection of chemical andbiological warfare weapons, and the basic research market sincefundamental cell processes, such as cell division and motility, arehighly dependent upon microtubule dynamics.

[0301] Image Acquisition and Analysis

[0302] Image data can be obtained from either fixed or living indicatorcells. To extract morphometric data from each of the images obtained thefollowing method of analysis is used:

[0303] 1. Threshold each nucleus and cytoplasmic image to produce a maskthat has value=0 for each pixel outside a nucleus or cell boundary.

[0304] 2. Overlay the mask on the original image, detect each object inthe field (i.e., nucleus or cell), and calculate its size, shape, andintegrated intensity.

[0305] 3. Overlay the whole cell mask obtained above on thecorresponding luminescent microtubule image and apply one or more of thefollowing set of classifiers to determine the micrtotubule morphologyand the effect of drugs on microtubule morphology.

[0306] Microtubule morphology is defined using a set of classifiers toquantify aspects of microtubule shape, size, aggregation state, andpolymerization state. These classifiers can be based on approaches thatinclude co-occurrence matrices, texture measurements, spectral methods,structural methods, wavelet transforms, statistical methods, orcombinations thereof. Examples of such classifiers are as follows:

[0307] 1. A classifier to quantify microtubule length and width usingedge detection methods such as that discussed in Kolega et al. ((1993).BioImaging 1:136-150), which discloses a non-automated method todetermine edge strength in individual cells), to calculate the totaledge strength within each cell. To normalize for cell size, the totaledge strength can be divided by the cell area to give a “microtubulemorphology” value. Large microtubule morphology values are associatedwith strong edge strength values and are therefore maximal in cellscontaining distinct microtubule structures. Likewise, small microtubulemorphology values are associated with weak edge strength and are minimalin cells with depolymerized microtubules. The physiological range ofmicrotubule morphology values is set by treating cells with either themicrotubule stabilizing drug paclitaxel (10 μM) or the microtubuledepolymerizing drug nocodazole (10 μg/ml).

[0308] 2. A classifier to quantify microtubule aggregation into punctatespots or foci using methodology from the receptor internalizationmethods discussed supra.

[0309] 3. A classifier to quantify microtubule depolymerization using ameasure of image texture.

[0310] 4. A classifier to quantify apparent interconnectivity, orbranching (or both), of the microtubules.

[0311] 5. Measurement of the kinetics of microtubule reorganizationusing the above classifiers on a time series of images of cells treatedwith test compounds.

[0312] In a further aspect, kits are provided for analyzing microtubulestability, comprising an expression vector comprising a nucleic acidthat encodes a microtubule labeling protein and instructions for usingthe expression vector for carrying out the methods described above. In apreferred embodiment, the expression vector further comprises a nucleicacid that encodes a luminescent protein, wherein the microtubule bindingprotein and the luminescent protein thereof are expressed as a fusionprotein. Alternatively, the kit may contain an antibody thatspecifically binds to the microtubule-labeling protein. In a furtherembodiment, the kit includes cells that express the microtubule labelingprotein. In a preferred embodiment, the cells are transfected with theexpression vector. In another preferred embodiment, the kits furthercontain a compound that is known to disrupt microtubule structure,including but not limited to curacin, nocodazole, vincristine, orvinblastine. In another preferred embodiment, the kits further comprisea compound that is known to stabilize microtubule structure, includingbut not limited to taxol (paclitaxel), and discodermolide.

[0313] In another aspect, the present invention comprises a machinereadable storage medium comprising a program containing a set ofinstructions for causing a cell screening system to execute thedisclosed methods for analyzing microtubule stability, wherein the cellscreening system comprises an optical system with a stage adapted forholding a plate containing cells, a digital camera, a means fordirecting fluorescence or luminescence emitted from the cells to thedigital camera, and a computer means for receiving and processing thedigital data from the digital camera.

[0314] High-Content Screens Involving the Functional Localization ofMacromolecules

[0315] Within this class of high-content screen, the functionallocalization of macromolecules in response to external stimuli ismeasured within living cells.

[0316] Glycolytic enzyme activity regulation. In a preferred embodimentof a cellular enzyme activity high-content screen, the activity of keyglycolytic regulatory enzymes are measured in treated cells. To measureenzyme activity, indicator cells containing luminescent labelingreagents are treated with test compounds and the activity of thereporters is measured in space and time using cell screening system ofthe present invention.

[0317] In one embodiment, the reporter of intracellular enzyme activityis fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase (PFK-2), aregulatory enzyme whose phosphorylation state indicates intracellularcarbohydrate anabolism or catabolism (Deprez et al. (1997) J. Biol.Chem. 272:17269-17275; Kealer et al. (1996) FEBS Letters 395:225-227;Lee et al. (1996), Biochemistry 35:6010-6019). The indicator cellscontain luminescent reporters consisting of a fluorescent proteinbiosensor of PFK-2 phosphorylation. The fluorescent protein biosensor isconstructed by introducing an environmentally sensitive fluorescent dyenear to the known phosphorylation site of the enzyme (Deprez et al.(1997), supra; Giuliano et al. (1995), supra). The dye can be of theketocyanine class (Kessler and Wolfbeis (1991), Spectrochimica Acta47A:187-192) or any class that contains a protein reactive moiety and afluorochrome whose excitation or emission spectrum is sensitive tosolution polarity. The fluorescent protein biosensor is introduced intothe indicator cells using bulk loading methodology.

[0318] Living indicator cells are treated with test compounds, at finalconcentrations ranging from 10⁻¹² M to 10⁻³ M for times ranging from 0.1s to 10 h. In a preferred embodiment, ratio image data are obtained fromliving treated indicator cells by collecting a spectral pair offluorescence images at each time point. To extract morphometric datafrom each time point, a ratio is made between each pair of images bynumerically dividing the two spectral images at each time point, pixelby pixel. Each pixel value is then used to calculate the fractionalphosphorylation of PFK-2. At small fractional values of phosphorylation,PFK-2 stimulates carbohydrate catabolism. At high fractional values ofphosphorylation, PFK-2 stimulates carbohydrate anabolism.

[0319] Protein kinase A activity and localization of subunits. Inanother embodiment of a high-content screen, both the domainlocalization and activity of protein kinase A (PKA) within indicatorcells are measured in response to treatment with test compounds.

[0320] The indicator cells contain luminescent reporters including afluorescent protein biosensor of PKA activation. The fluorescent proteinbiosensor is constructed by introducing an environmentally sensitivefluorescent dye into the catalytic subunit of PKA near the site known tointeract with the regulatory subunit of PKA (Harootunian et al. (1993),Mol. Biol. of the Cell 4:993-1002; Johnson et al. (1996), Cell85:149-158; Giuliano et al. (1995), supra). The dye can be of theketocyanine class (Kessler, and Wolfbeis (1991), Spectrochimica Acta47A: 187-192) or any class that contains a protein reactive moiety and afluorochrome whose excitation or emission spectrum is sensitive tosolution polarity. The fluorescent protein biosensor of PKA activationis introduced into the indicator cells using bulk loading methodology.

[0321] In one embodiment, living indicator cells are treated with testcompounds, at final concentrations ranging from 10⁻¹² M to 10⁻³ M fortimes ranging from 0.1 s to 10 h. In a preferred embodiment, ratio imagedata are obtained from living treated indicator cells. To extractbiosensor data from each time point, a ratio is made between each pairof images, and each pixel value is then used to calculate the fractionalactivation of PKA (e.g., separation of the catalytic and regulatorysubunits after cAMP binding). At high fractional values of activity,PFK-2 stimulates biochemical cascades within the living cell.

[0322] To measure the translocation of the catalytic subunit of PKA,indicator cells containing luminescent reporters are treated with testcompounds and the movement of the reporters is measured in space andtime using the cell screening system. The indicator cells containluminescent reporters consisting of domain markers used to measure thelocalization of the cytoplasmic and nuclear domains. When the indicatorcells are treated with a test compounds, the dynamic redistribution of aPKA fluorescent protein biosensor is recorded intracellularly as aseries of images over a time scale ranging from 0.1 s to 10 h. Eachimage is analyzed by a method that quantifies the movement of the PKAbetween the cytoplasmic and nuclear domains. To do this calculation, theimages of the probes used to mark the cytoplasmic and nuclear domainsare used to mask the image of the PKA fluorescent protein biosensor. Theintegrated brightness per unit area under each mask is used to form atranslocation quotient by dividing the cytoplasmic integratedbrightness/area by the nuclear integrated brightness/area. By comparingthe translocation quotient values from control and experimental wells,the percent translocation is calculated for each potential leadcompound. The output of the high-content screen relates quantitativedata describing the magnitude of the translocation within a large numberof individual cells that have been treated with test compound in theconcentration range of 10⁻¹² M to 10⁻³ M.

[0323] High-Content Screens Involving the Induction or Inhibition ofGene Expression RNA-Based Fluorescent Biosensors

[0324] Cytoskeletal protein transcription and message localization.Regulation of the general classes of cell physiological responsesincluding cell-substrate adhesion, cell-cell adhesion, signaltransduction, cell-cycle events, intermediary and signaling moleculemetabolism, cell locomotion, cell-cell communication, and cell death caninvolve the alteration of gene expression. High-content screens can alsobe designed to measure this class of physiological response.

[0325] In one embodiment, the reporter of intracellular gene expressionis an oligonucleotide that can hybridize with the target mRNA and alterits fluorescence signal. In a preferred embodiment, the oligonucleotideis a molecular beacon (Tyagi and Kramer (1996) Nat. Biotechnol.14:303-308), a luminescence-based reagent whose fluorescence signal isdependent on intermolecular and intramolecular interactions. Thefluorescent biosensor is constructed by introducing a fluorescenceenergy transfer pair of fluorescent dyes such that there is one at eachend (5′ and 3′) of the reagent. The dyes can be of any class thatcontains a protein reactive moiety and fluorochromes whose excitationand emission spectra overlap sufficiently to provide fluorescence energytransfer between the dyes in the resting state, including, but notlimited to, fluorescein and rhodamine (Molecular Probes, Inc.). In apreferred embodiment, a portion of the message coding for β-actin(Kislauskis et al. (1994), J. Cell Biol. 127:441-451; McCann et al.(1997), Proc. Natl. Acad. Sci. 94:5679-5684; Sutoh (1982), Biochemistry21:3654-3661) is inserted into the loop region of a hairpin-shapedoligonucleotide with the ends tethered together due to intramolecularhybridization. At each end of the biosensor a fluorescence donor(fluorescein) and a fluorescence acceptor (rhodamine) are covalentlybound. In the tethered state, the fluorescence energy transfer ismaximal and therefore indicative of an unhybridized molecule. Whenhybridized with the mRNA coding for β-actin, the tether is broken andenergy transfer is lost. The complete fluorescent biosensor isintroduced into the indicator cells using bulk loading methodology.

[0326] In one embodiment, living indicator cells are treated with testcompounds, at final concentrations ranging from 10⁻¹² M to 10⁻³ M fortimes ranging from 0.1 s to 10 h. In a preferred embodiment, ratio imagedata are obtained from living treated indicator cells. To extractmorphometric data from each time point, a ratio is made between eachpair of images, and each pixel value is then used to calculate thefractional hybridization of the labeled nucleotide. At small fractionalvalues of hybridization little expression of β-actin is indicated. Athigh fractional values of hybridization, maximal expression of β-actinis indicated. Furthermore, the distribution of hybridized moleculeswithin the cytoplasm of the indicator cells is also a measure of thephysiological response of the indicator cells.

[0327] Cell Surface Binding of a Ligand

[0328] Labeled insulin binding to its cell surface receptor in livingcells. Cells whose plasma membrane domain has been labeled with alabeling reagent of a particular color are incubated with a solutioncontaining insulin molecules (Lee et al. (1997), Biochemistry36:2701-2708; Martinez-Zaguilan et al. (1996), Am. J. Physiol.270:C1438-C1446) that are labeled with a luminescent probe of adifferent color for an appropriate time under the appropriateconditions. After incubation, unbound insulin molecules are washed away,the cells fixed and the distribution and concentration of the insulin onthe plasma membrane is measured. To do this, the cell membrane image isused as a mask for the insulin image. The integrated intensity from themasked insulin image is compared to a set of images containing knownamounts of labeled insulin. The amount of insulin bound to the cell isdetermined from the standards and used in conjunction with the totalconcentration of insulin incubated with the cell to calculate adissociation constant or insulin to its cell surface receptor.

[0329] Labeling of Cellular Compartments

[0330] Whole Cell Labeling

[0331] Whole cell labeling is accomplished by labeling cellularcomponents such that dynamics of cell shape and motility of the cell canbe measured over time by analyzing fluorescence images of cells.

[0332] In one embodiment, small reactive fluorescent molecules areintroduced into living cells. These membrane-permeant molecules bothdiffuse through and react with protein components in the plasmamembrane. Dye molecules react with intracellular molecules to bothincrease the fluorescence signal emitted from each molecule and toentrap the fluorescent dye within living cells. These molecules includereactive chloromethyl derivatives of aminocoumarins, hydroxycoumarins,eosin diacetate, fluorescein diacetate, some Bodipy dye derivatives, andtetramethylrhodamine. The reactivity of these dyes toward macromoleculesincludes free primary amino groups and free sulfhydryl groups.

[0333] In another embodiment, the cell surface is labeled by allowingthe cell to interact with fluorescently labeled antibodies or lectins(Sigma Chemical Company, St. Louis, Mo.) that react specifically withmolecules on the cell surface. Cell surface protein chimeras expressedby the cell of interest that contain a green fluorescent protein, ormutant thereof, component can also be used to fluorescently label theentire cell surface. Once the entire cell is labeled, images of theentire cell or cell array can become a parameter in high contentscreens, involving the measurement of cell shape, motility, size, andgrowth and division.

[0334] Plasma Membrane labeling

[0335] In one embodiment, labeling the whole plasma membrane employssome of the same methodology described above for labeling the entirecells. Luminescent molecules that label the entire cell surface act todelineate the plasma membrane.

[0336] In a second embodiment subdomains of the plasma membrane, theextracellular surface, the lipid bilayer, and the intracellular surfacecan be labeled separately and used as components of high contentscreens. In the first embodiment, the extracellular surface is labeledusing a brief treatment with a reactive fluorescent molecule such as thesuccinimidyl ester or iodoacetamde derivatives of fluorescent dyes suchas the fluoresceins, rhodamines, cyanines, and Bodipys.

[0337] In a third embodiment, the extracellular surface is labeled usingfluorescently labeled macromolecules with a high affinity for cellsurface molecules. These include fluorescently labeled lectins such asthe fluorescein, rhodamine, and cyanine derivatives of lectins derivedfrom jack bean (Con A), red kidney bean (erythroagglutinin PHA-E), orwheat germ.

[0338] In a fourth embodiment, fluorescently labeled antibodies with ahigh affinity for cell surface components are used to label theextracellular region of the plasma membrane. Extracellular regions ofcell surface receptors and ion channels are examples of proteins thatcan be labeled with antibodies.

[0339] In a fifth embodiment, the lipid bilayer of the plasma membraneis labeled with fluorescent molecules. These molecules includefluorescent dyes attached to long chain hydrophobic molecules thatinteract strongly with the hydrophobic region in the center of theplasma membrane lipid bilayer. Examples of these dyes include the PKHseries of dyes (U.S. Pat. Nos. 4,783,401, 4,762701, and 4,859,584;available commercially from Sigma Chemical Company, St. Loius, Mo.),fluorescent phospholipids such as nitrobenzoxadiazoleglycerophosphoethanolamine and fluorescein-derivatizeddihexadecanoylglycerophosphoethanolamine, fluorescent fatty acids suchas 5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonanoic acidand 1-pyrenedecanoic acid (Molecular Probes, Inc.), fluorescent sterolsincluding cholesteryl4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoateand cholesteryl 1-pyrenehexanoate, and fluorescently labeled proteinsthat interact specifically with lipid bilayer components such as thefluorescein derivative of annexin V (Caltag Antibody Co, Burlingame,Calif.).

[0340] In another embodiment, the intracellular component of the plasmamembrane is labeled with fluorescent molecules. Examples of thesemolecules are the intracellular components of the trimeric G-proteinreceptor, adenylyl cyclase, and ionic transport proteins. Thesemolecules can be labeled as a result of tight binding to a fluorescentlylabeled specific antibody or by the incorporation of a fluorescentprotein chimera that is comprised of a membrane-associated protein andthe green fluorescent protein, and mutants thereof.

[0341] Endosome Fluorescence Labeling

[0342] In one embodiment, ligands that are transported into cells byreceptor-mediated endocytosis are used to trace the dynamics ofendosomal organelles. Examples of labeled ligands include BodipyFL-labeled low density lipoprotein complexes, tetramethylrhodaminetransferrin analogs, and fluorescently labeled epidermal growth factor(Molecular Probes, Inc.)

[0343] In a second embodiment, fluorescently labeled primary orsecondary antibodies (Sigma Chemical Co. St. Louis, Mo.; MolecularProbes, Inc. Eugene, Oreg.; Caltag Antibody Co.) that specifically labelendosomal ligands are used to mark the endosomal compartment in cells.

[0344] In a third embodiment, endosomes are fluorescently labeled incells expressing protein chimeras formed by fusing a green fluorescentprotein, or mutants thereof, with a receptor whose internalizationlabels endosomes. Chimeras of the EGF, transferrin, and low densitylipoprotein receptors are examples of these molecules.

[0345] Lysosome Labeling

[0346] In one embodiment, membrane permeant lysosome-specificluminescent reagents are used to label the lysosomal compartment ofliving and fixed cells. These reagents include the luminescent moleculesneutral red,N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine, andthe LysoTracker probes which report intralysosomal pH as well as thedynamic distribution of lysosomes (Molecular Probes, Inc.)

[0347] In a second embodiment, antibodies against lysosomal antigens(Sigma Chemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) areused to label lysosomal components that are localized in specificlysosomal domains. Examples of these components are the degradativeenzymes involved in cholesterol ester hydrolysis, membrane proteinproteases, and nucleases as well as the ATP-driven lysosomal protonpump.

[0348] In a third embodiment, protein chimeras consisting of a lysosomalprotein genetically fused to an intrinsically luminescent protein suchas the green fluorescent protein, or mutants thereof, are used to labelthe lysosomal domain. Examples of these components are the degradativeenzymes involved in cholesterol ester hydrolysis, membrane proteinproteases, and nucleases as well as the ATP-driven lysosomal protonpump.

[0349] Cytoplasmic Fluorescence Labeling

[0350] In one embodiment, cell permeant fluorescent dyes (MolecularProbes, Inc.) with a reactive group are reacted with living cells.Reactive dyes including monobromobimane, 5-chloromethylfluoresceindiacetate, carboxy fluorescein diacetate succinimidyl ester, andchloromethyl tetramethylrhodamine are examples of cell permeantfluorescent dyes that are used for long term labeling of the cytoplasmof cells.

[0351] In a second embodiment, polar tracer molecules such as Luciferyellow and cascade blue-based fluorescent dyes (Molecular Probes, Inc.)are introduced into cells using bulk loading methods and are also usedfor cytoplasmic labeling.

[0352] In a third embodiment, antibodies against cytoplasmic components(Sigma Chemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) areused to fluorescently label the cytoplasm. Examples of cytoplasmicantigens are many of the enzymes involved in intermediary metabolism.Enolase, phosphofructokinase, and acetyl-CoA dehydrogenase are examplesof uniformly distributed cytoplasmic antigens.

[0353] In a fourth embodiment, protein chimeras consisting of acytoplasmic protein genetically fused to an intrinsically luminescentprotein such as the green fluorescent protein, or mutants thereof, areused to label the cytoplasm. Fluorescent chimeras of uniformlydistributed proteins are used to label the entire cytoplasmic domain.Examples of these proteins are many of the proteins involved inintermediary metabolism and include enolase, lactate dehydrogenase, andhexokinase.

[0354] In a fifth embodiment, antibodies against cytoplasmic antigens(Sigma Chemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) areused to label cytoplasmic components that are localized in specificcytoplasmic sub-domains. Examples of these components are thecytoskeletal proteins actin, tubulin, and cytokeratin. A population ofthese proteins within cells is assembled into discrete structures, whichin this case, are fibrous. Fluorescence labeling of these proteins withantibody-based reagents therefore labels a specific sub-domain of thecytoplasm.

[0355] In a sixth embodiment, non-antibody-based fluorescently labeledmolecules that interact strongly with cytoplasmic proteins are used tolabel specific cytoplasmic components. One example is a fluorescentanalog of the enzyme DNAse I (Molecular Probes, Inc.) Fluorescentanalogs of this enzyme bind tightly and specifically to cytoplasmicactin, thus labeling a sub-domain of the cytoplasm. In another example,fluorescent analogs of the mushroom toxin phalloidin or the drugpaclitaxel (Molecular Probes, Inc.) are used to label components of theactin- and microtubule-cytoskeletons, respectively.

[0356] In a seventh embodiment, protein chimeras consisting of acytoplasmic protein genetically fused to an intrinsically luminescentprotein such as the green fluorescent protein, or mutants thereof, areused to label specific domains of the cytoplasm. Fluorescent chimeras ofhighly localized proteins are used to label cytoplasmic sub-domains.Examples of these proteins are many of the proteins involved inregulating the cytoskeleton. They include the structural proteins actin,tubulin, and cytokeratin as well as the regulatory proteins microtubuleassociated protein 4 and α-actinin.

[0357] Nuclear labeling

[0358] In one embodiment, membrane permeant nucleic-acid-specificluminescent reagents (Molecular Probes, Inc.) are used to label thenucleus of living and fixed cells. These reagents include cyanine-baseddyes (e.g., TOTO®, YOYO®, and BOBO™), phenanthidines and acridines(e.g., ethidium bromide, propidium iodide, and acridine orange), indolesand imidazoles (e.g., Hoechst 33258, Hoechst 33342, and4′,6-diamidino-2-phenylindole), and other similar reagents (e.g.,7-aminoactinomycin D, hydroxystilbamidine, and the psoralens).

[0359] In a second embodiment, antibodies against nuclear antigens(Sigma Chemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) areused to label nuclear components that are localized in specific nucleardomains. Examples of these components are the macromolecules involved inmaintaining DNA structure and function. DNA, RNA, histones, DNApolymerase, RNA polymerase, lamins, and nuclear variants of cytoplasmicproteins such as actin are examples of nuclear antigens.

[0360] In a third embodiment, protein chimeras consisting of a nuclearprotein genetically fused to an intrinsically luminescent protein suchas the green fluorescent protein, or mutants thereof, are used to labelthe nuclear domain. Examples of these proteins are many of the proteinsinvolved in maintaining DNA structure and function. Histones, DNApolymerase, RNA polymerase, lamins, and nuclear variants of cytoplasmicproteins such as actin are examples of nuclear proteins.

[0361] Mitochondrial Labeling

[0362] In one embodiment, membrane permeant mitochondrial-specificluminescent reagents (Molecular Probes, Inc.) are used to label themitochondria of living and fixed cells. These reagents include rhodamine123, tetramethyl rosamine, JC-1, and the MitoTracker reactive dyes.

[0363] In a second embodiment, antibodies against mitochondrial antigens(Sigma Chemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) areused to label mitochondrial components that are localized in specificmitochondrial domains. Examples of these components are themacromolecules involved in maintaining mitochondrial DNA structure andfunction. DNA, RNA, histones, DNA polymerase, RNA polymerase, andmitochondrial variants of cytoplasmic macromolecules such asmitochondrial tRNA and rRNA are examples mitochondrial antigens. Otherexamples of mitochondrial antigens are the components of the oxidativephosphorylation system found in the mitochondria (e.g., cytochrome c,cytochrome c oxidase, and succinate dehydrogenase).

[0364] In a third embodiment, protein chimeras consisting of amitochondrial protein genetically fused to an intrinsically luminescentprotein such as the green fluorescent protein, or mutants thereof, areused to label the mitochondrial domain. Examples of these components arethe macromolecules involved in maintaining mitochondrial DNA structureand function. Examples include histones, DNA polymerase, RNA polymerase,and the components of the oxidative phosphorylation system found in themitochondria (e.g., cytochrome c, cytochrome c oxidase, and succinatedehydrogenase).

[0365] Endoplasmic Reticulum Labeling

[0366] In one embodiment, membrane permeant endoplasmicreticulum-specific luminescent reagents (Molecular Probes, Inc.) areused to label the endoplasmic reticulum of living and fixed cells. Thesereagents include short chain carbocyanine dyes (e.g., DiOC₆ and DiOC₃),long chain carbocyanine dyes (e.g., DiIC₁₆ and DiIC₈), and luminescentlylabeled lectins such as concanavalin A.

[0367] In a second embodiment, antibodies against endoplasmic reticulumantigens (Sigma Chemical Co.; Molecular Probes, Inc.; Caltag AntibodyCo.) are used to label endoplasmic reticulum components that arelocalized in specific endoplasmic reticulum domains. Examples of thesecomponents are the macromolecules involved in the fatty acid elongationsystems, glucose-6-phosphatase, and HMG CoA-reductase.

[0368] In a third embodiment, protein chimeras consisting of aendoplasmic reticulum protein genetically fused to an intrinsicallyluminescent protein such as the green fluorescent protein, or mutantsthereof, are used to label the endoplasmic reticulum domain. Examples ofthese components are the macromolecules involved in the fatty acidelongation systems, glucose-6-phosphatase, and HMG CoA-reductase.

[0369] Golgi Labeling

[0370] In one embodiment, membrane permeant Golgi-specific luminescentreagents (Molecular Probes, Inc.) are used to label the Golgi of livingand fixed cells. These reagents include luminescently labeledmacromolecules such as wheat germ agglutinin and Brefeldin A as well asluminescently labeled ceramide.

[0371] In a second embodiment, antibodies against Golgi antigens (SigmaChemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) are used tolabel Golgi components that are localized in specific Golgi domains.Examples of these components are N-acetylglucosamine phosphotransferase,Golgi-specific phosphodiesterase, and mannose-6-phosphate receptorprotein.

[0372] In a third embodiment, protein chimeras consisting of a Golgiprotein genetically fused to an intrinsically luminescent protein suchas the green fluorescent protein, or mutants thereof, are used to labelthe Golgi domain. Examples of these components are N-acetylglucosaminephosphotransferase, Golgi-specific phosphodiesterase, andmannose-6-phosphate receptor protein.

[0373] While many of the examples presented involve the measurement ofsingle cellular processes, this is again is intended for purposes ofillustration only. Multiple parameter high-content screens can beproduced by combining several single parameter screens into amultiparameter high-content screen or by adding cellular parameters toany existing high-content screen. Furthermore, while each example isdescribed as being based on either live or fixed cells, eachhigh-content screen can be designed to be used with both live and fixedcells.

[0374] Those skilled in the art will recognize a wide variety ofdistinct screens that can be developed based on the disclosure providedherein. There is a large and growing list of known biochemical andmolecular processes in cells that involve translocations orreorganizations of specific components within cells. The signalingpathway from the cell surface to target sites within the cell involvesthe translocation of plasma membrane-associated proteins to thecytoplasm. For example, it is known that one of the src family ofprotein tyrosine kinases, pp60c-src (Walker et al (1993), J. Biol. Chem.268:19552-19558) translocates from the plasma membrane to the cytoplasmupon stimulation of fibroblasts with platelet-derived growth factor(PDGF). Additionally, the targets for screening can themselves beconverted into fluorescence-based reagents that report molecular changesincluding ligand-binding and post-translocational modifications.

EXAMPLE 10 Protease Biosensors

[0375] (1) Background

[0376] As used herein, the following terms are defined as follows:

[0377] Reactant—the parent biosensor that interacts with the proteolyticenzyme.

[0378] Product—the signal-containing proteolytic fragment(s) generatedby the interaction of the reactant with the enzyme.

[0379] Reactant Target Sequence—an amino acid sequence that imparts arestriction on the cellular distribution of the reactant to a particularsubcellular domain of the cell.

[0380] Product Target Sequence—an amino acid sequence that imparts arestriction on the cellular distribution of the signal-containingproduct(s) of the targeted enzymatic reaction to a particularsubcellular domain of the cell. If the product is initially localizedwithin a membrane bound compartment, then the Product Target Sequencemust incorporate the ability to export the product out of themembrane-bound compartment. A bi-functional sequence can be used, whichfirst moves the product out of the membrane-bound compartment, and thentargets the product to the final compartment. In general, the same aminoacid sequences can act as either or both reactant target sequences andproduct target sequences. Exceptions to this include amino acidsequences which target the nuclear is envelope, Golgi apparatus,endoplasmic reticuulum, and which are involved in famesylation, whichare more suitable as reactant target sequences.

[0381] Protease Recognition Site—an amino acid sequence that impartsspecificity by mimicking the substrate, providing a specific binding andcleavage site for a protease. Although typically a short sequence ofamino acids representing the minimal cleavage site for a protease (e.g.DEVD for caspase-3, Villa, P., S. H. Kaufmann, and W. C. Earnshaw. 1997.Caspases and caspase inhibitors. Trends Biochem Sci. 22:388-93), greaterspecificity may be established by using a longer sequence from anestablished substrate.

[0382] Compartment—any cellular sub-structure or macromolecularcomponent of the cell, whether it is made of protein, lipid,carbohydrate, or nucleic acid. It could be a macromolecular assembly oran organelle (a membrane delimited cellular component). Compartmentsinclude, but are not limited to, cytoplasm, nucleus, nucleolus, innerand outer surface of nuclear envelope, cytoskeleton, peroxisome,endosome, lysosome, inner leaflet of plasma membrane, outer leaflet ofplasma membrane, outer leaflet of mitochondrial membrane, inner leafletof mitochondrial membrane, Golgi, endoplasmic reticulum, orextracellular space.

[0383] Signal—an amino acid sequence that can be detected. Thisincludes, but is not limited to inherently fluorescent proteins (e.g.Green Fluorescent Protein), cofactor-requiring fluorescent orluminescent proteins (e.g. phycobiliproteins or luciferases), andepitopes recognizable by specific antibodies or other specific naturalor unnatural binding probes, including but not limited to dyes, enzymecofactors and engineered binding molecules, which are fluorescently orluminescently labeled. Also included are site-specifically labeledproteins that contain a luminescent dye. Methodology for site-specificlabeling of proteins includes, but is not limited to, engineereddye-reactive amino acids (Post, et al., J. Biol. Chem. 269:12880-12887(1994)), enzyme-based incorporation of luminescent substrates intoproteins (Buckler, et al., Analyt. Biochem. 209:20-31 (1993); Takashi,Biochemistry. 27:938-943 (1988)), and the incorporation of unnaturallabeled amino acids into proteins (Noren, et al., Science. 244:182-188(1989)).

[0384] Detection—a means for recording the presence, position, or amountof the signal. The approach may be direct, if the signal is inherentlyfluorescent, or indirect, if, for example, the signal is an epitope thatmust be subsequently detected with a labeled antibody. Modes ofdetection include, but are not limited to, the spatial position offluorescence, luminescence, or phosphorescence: (1) intensity; (2)polarization; (3) lifetime; (4) wavelength; (5) energy transfer; and (6)recovery after photobleaching.

[0385] The basic principle of the protease biosensors of the presentinvention is to spatially separate the reactants from the productsgenerated during a proteolytic reaction. The separation of products fromreactants occurs upon proteolytic cleavage of the protease recognitionsite within the biosensor, allowing the products to bind to, diffuseinto, or be imported into compartments of the cell different from thoseof the reactant. This spatial separation provides a means ofquantitating a proteolytic process directly in living or fixed cells.Some designs of the biosensor provide a means of restricting thereactant (uncleaved biosensor) to a particular compartment by a proteinsequence (“reactant target sequence”) that binds to or imports thebiosensor into a compartment of the cell. These compartments include,but are not limited to any cellular substructure, macromolecularcellular component, membrane-limited organelles, or the extracellularspace. Given that the characteristics of the proteolytic reaction arerelated to product concentration divided by the reactant concentration,the spatial separation of products and reactants provides a means ofuniquely quantitating products and reactants in single cells, allowing amore direct measure of proteolytic activity.

[0386] The molecular-based biosensors may be introduced into cells viatransfection and the expressed chimeric proteins analyzed in transientcell populations or stable cell lines. They may also be pre-formed, forexample by production in a prokaryotic or eukaryotic expression system,and the purified protein introduced into the cell via a number ofphysical mechanisms including, but not limited to, micro-injection,scrape loading, electroporation, signal-sequence mediated loading, etc.

[0387] Measurement modes may include, but are not limited to, the ratioor difference in fluorescence, luminescence, or phosphorescence: (a)intensity; (b) polarization; or (c) lifetime between reactant andproduct. These latter modes require appropriate spectroscopicdifferences between products and reactants. For example, cleaving areactant containing a limited-mobile signal into a very smalltranslocating component and a relatively large non-translocatingcomponent may be detected by polarization. Alternatively, significantlydifferent emission lifetimes between reactants and products allowdetection in imaging and non-imaging modes.

[0388] One example of a family of enzymes for which this biosensor canbe constructed to report activity is the caspases. Caspases are a classof proteins that catalyze proteolytic cleavage of a wide variety oftargets during apoptosis. Following initiation of apoptosis, the ClassII “downstream” caspases are activated and are the point of no return inthe pathway leading to cell death, resulting in cleavage of downstreamtarget proteins. In specific examples, the biosensors described herewere engineered to use nuclear translocation of cleaved GFP as ameasurable indicator of caspase activation. Additionally, the use ofspecific recognition sequences that incorporate surrounding amino acidsinvolved in secondary structure formation in naturally occurringproteins may increase the specificity and sensitivity of this class ofbiosensor.

[0389] Another example of a protease class for which this biosensor canbe constructed to report activity is zinc metalloproteases. Two specificexamples of this class are the biological toxins derived fromClostridial species (C. botulinum and C. tetani) and Bacillus anthracis.(Herreros et al. In The Comprehensive Sourcebook of Bacterial ProteinToxins. J. E. Alouf and J. H. Freer, Eds. 2^(nd) edition, San Diego,Academic Press, 1999; pp 202-228.) These bacteria express and secretezinc metalloproteases that enter eukaryotic cells and specificallycleave distinct target proteins. For example, the anthrax protease fromBacillus anthracis is delivered into the cytoplasm of target cells viaan accessory pore-forming protein, where its proteolytic activityinactivates the MAP-kinase signaling cascade through cleavage of mitogenactivated protein kinase kinases 1 or 2 (MEK1 or MEK2). (Leppla, S. A.In The Comprehensive Sourcebook of Bacterial Protein Toxins. J. E. Aloufand J. H. Freer, Eds. 2^(nd) edition, San Diego, Academic Press, 1999;pp243-263.) The toxin biosensors described here take advantage of thenatural subcellular localization of these and other target proteins toachieve reactant targeting. Upon cleavage, the signal (with or without aproduct target sequence) is separated from the reactant to create ahigh-content biosensor.

[0390] One of skill in the art will recognize that the proteinbiosensors of this aspect of the invention can be adapted to report theactivity of any member of the caspase family of proteases, as well asany other protease, by a substitution of the appropriate proteaserecognition site in any of the constructs (see FIG. 29B). Thesebiosensors can be used in high-content screens to detect in vivoactivation of enzymatic activity and to identify specific activity basedon cleavage of a known recognition motif. This screen can be used forboth live cell and fixed end-point assays, and can be combined withadditional measurements to provide a multi-parameter assay.

[0391] Thus, in another aspect the present invention providesrecombinant nucleic acids encoding a protease biosensor, comprising:

[0392] a. a first nucleic acid sequence that encodes at least onedetectable polypeptide signal;

[0393] b. a second nucleic acid sequence that encodes at least oneprotease recognition site, wherein the second nucleic acid sequence isoperatively linked to the first nucleic acid sequence that encodes theat least one detectable polypeptide signal; and

[0394] c. a third nucleic acid sequence that encodes at least onereactant target sequence, wherein the third nucleic acid sequence isoperatively linked to the second nucleic acid sequence that encodes theat least one protease recognition site.

[0395] In this aspect, the first and third nucleic acid sequences areseparated by the second nucleic acid sequence, which encodes theprotease recognition site.

[0396] In a further embodiment, the recombinant nucleic acid encoding aprotease biosensor comprises a fourth nucleic acid sequence that encodesat least one product target sequence, wherein the fourth nucleic acidsequence is operatively linked to the first nucleic acid sequence thatencodes the at least one detectable polypeptide signal.

[0397] In a further embodiment, the recombinant nucleic acid encoding aprotease biosensor comprises a fifth nucleic acid sequence that encodesat least one detectable polypeptide signal, wherein the fifth nucleicacid sequence is operatively linked to the third nucleic acid sequencethat encodes the reactant target sequence.

[0398] In a preferred embodiment, the detectable polypeptide signal isselected from the group consisting of fluorescent proteins, luminescentproteins, and sequence epitopes. In a most preferred embodiment, thefirst nucleic acid encoding a polypeptide sequence comprises a sequenceselected from the group consisting of SEQ ID NOS: 35, 37, 39, 41, 43,45, 47, 49, and 51.

[0399] In another preferred embodiment, the second nucleic acid encodinga protease recognition site comprises a sequence selected from the groupconsisting of SEQ ID NOS: 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, and 121. In another preferred embodiment,the third nucleic acid encoding a reactant target sequence comprises asequence selected from the group consisting of SEQ ID NOS: 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, and 151.

[0400] In a most preferred embodiment, the recombinant nucleic acidencoding a protease biosensor comprises a sequence substantially similarto sequences selected from the group consisting of SEQ ID NOS:1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.

[0401] In another aspect, the present invention provides a recombinantexpression vector comprising nucleic acid control sequences operativelylinked to the above-described recombinant nucleic acids. In a stillfurther aspect, the present invention provides genetically engineeredhost cells that have been transfected with the recombinant expressionvectors of the invention.

[0402] In another aspect, the present invention provides recombinantprotease biosensors comprising

[0403] a. a first domain comprising at least one detectable polypeptidesignal;

[0404] b. a second domain comprising at least one protease recognitionsite; and

[0405] c. a third domain comprising at least one reactant targetsequence;

[0406] wherein the first domain and the third domain are separated bythe second domain.

[0407] Inherent in this embodiment is the concept that the reactanttarget sequence restricts the cellular distribution of the reactant,with redistribution of the product occurring after activation (ie:protease cleavage). This redistribution does not require a completesequestration of products and reactants, as the product distribution canpartially overlap the reactant distribution in the absence of a producttargeting signal (see below).

[0408] In a preferred embodiment, the recombinant protease biosensorfurther comprises a fourth domain comprising at least one product targetsequence, wherein the fourth domain and the first domain are operativelylinked and are separated from the third domain by the second domain. Inanother embodiment, the recombinant protease biosensor further comprisesa fifth domain comprising at least one detectable polypeptide signal,wherein the fifth domain and the third domain are operatively linked andare separated from the first domain by the second domain.

[0409] In a preferred embodiment, the detectable polypeptide signaldomain (first or fifth domain) is selected from the group consisting offluorescent proteins, luminescent proteins, and sequence epitopes. In amost preferred embodiment, the detectable polypeptide signal domaincomprises a sequence selected from the group consisting of SEQ IDNOS:36, 38, 40, 42, 44, 46, 48, 50, and 52.

[0410] In another preferred embodiment, the second domain comprising aprotease recognition site comprises a sequence selected from the groupconsisting of SEQ ID NOS:54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, and 122. In another preferred embodiment,the reactant and/or target sequence domains comprise a sequence selectedfrom the group consisting of SEQ ID NOS:124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, and 152.

[0411] In a most preferred embodiment, the recombinant proteasebiosensor comprises a sequence substantially similar to sequencesselected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, and 34.

[0412] In a still further embodiment, the present invention providesmethods and kits for automated analysis of cells, comprising using cellsthat possess the protease biosensors of the invention to identifycompounds that affect protease activity. The method can be combined withthe other methods of the invention in a variety of possiblemulti-parametric assays.

[0413] In these various embodiments, the basic protease biosensor iscomposed of multiple domains, including at least a first detectablepolypeptide signal domain, at least one reactant target domain, and atleast one protease recognition domain, wherein the detectable signaldomain and the reactant target domain are separated by the proteaserecognition domain. Thus, the exact order of the domains in the moleculeis not generally critical, so long as the protease recognition domainseparates the reactant target and first detectable signal domain. Foreach domain, one or more one of the specified recognition sequences ispresent.

[0414] In some cases, the order of the domains in the biosensor may becritical for appropriate targeting of product(s) and/or reactant to theappropriate cellular compartment(s). For example, the targeting ofproducts or reactants to the peroxisome requires that the peroxisomaltargeting domain comprise the last three amino acids of the protein.Determination of those biosensor in which the relative placement oftargeting domains within the biosensor is critical can be determined byone of skill in the art through routine experimentation.

[0415] Some examples of the basic organization of domains within theprotease biosensor are shown in FIG. 30. One of skill in the art willrecognize that any one of a wide variety of protease recognition sites,product target sequences, polypeptide signals, and/or product targetsequences can be used in various combinations in the protein biosensorof the present invention, by substituting the appropriate codingsequences into the multi-domain construct. Non-limiting examples of suchalternative sequences are shown in FIGS. 29A-29C. Similarly, one ofskill in the art will recognize that modifications, substitutions, anddeletions can be made to the coding sequences and the amino acidsequence of each individual domain within the biosensor, while retainingthe function of the domain. Such various combinations of domains andmodifications, substitutions and deletions to individual domains arewithin the scope of the invention.

[0416] As used herein, the term “coding sequence” or a sequence which“encodes” a particular polypeptide sequence, refers to a nucleic acidsequence which is transcribed (in the case of DNA) and translated (inthe case of mRNA) into a polypeptide in vitro or in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Acoding sequence can include, but is not limited to, cDNA fromprokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryoticor eukaryotic DNA, and synthetic DNA sequences. A transcriptiontermination sequence will usually be located 3′ to the coding sequence.

[0417] As used herein, the term DNA “control sequences” referscollectively to promoter sequences, ribosome binding sites,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, enhancers, and the like, which collectively providefor the transcription and translation of a coding sequence in a hostcell. Not all of these control sequences need always be present in arecombinant vector so long as the DNA sequence of interest is capable ofbeing transcribed and translated appropriately.

[0418] As used herein, the term “operatively linked” refers to anarrangement of elements wherein the components so described areconfigured so as to perform their usual function. Thus, controlsequences operatively linked to a coding sequence are capable ofeffecting the expression of the coding sequence. The control sequencesneed not be contiguous with the coding sequence, so long as theyfunction to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operatively linked” to the codingsequence.

[0419] Furthermore, a nucleic acid coding sequence is operatively linkedto another nucleic acid coding sequences when the coding region for bothnucleic acid molecules are capable of expression in the same readingframe. The nucleic acid sequences need not be contiguous, so long asthey are capable of expression in the same reading frame. Thus, forexample, intervening coding regions can be present between the specifiednucleic acid coding sequences, and the specified nucleic acid codingregions can still be considered “operatively linked”.

[0420] The intervening coding sequences between the various domains ofthe biosensors can be of any length so long as the function of eachdomain is retained. Generally, this requires that the two-dimensionaland three-dimensional structure of the intervening protein sequence doesnot preclude the binding or interaction requirements of the domains ofthe biosensor, such as product or reactant targeting, binding of theprotease of interest to the biosensor, fluorescence or luminescence ofthe detectable polypeptide signal, or binding of fluorescently labeledepitope-specific antibodies. One case where the distance between domainsof the protease biosensor is important is where the goal is to create afluorescence resonance energy transfer pair. In this case, the FRETsignal will only exist if the distance between the donor and acceptor issufficiently small as to allow energy transfer (Tsien, Heim and Cubbit,WO 97/28261). The average distance between the donor and acceptormoieties should be between 1 nm and 10 nm with a preference of between 1nm and 6 nm. This is the physical distance between donor and acceptor.The intervening sequence length can vary considerably since the threedimensional structure of the peptide will determine the physicaldistance between donor and acceptor.

[0421] “Recombinant expression vector” includes vectors that operativelylink a nucleic acid coding region or gene to any promoter capable ofeffecting expression of the gene product. The promoter sequence used todrive expression of the protease biosensor may be constitutive (drivenby any of a variety of promoters, including but not limited to, CMV,SV40, RSV, actin, EF) or inducible (driven by any of a number ofinducible promoters including, but not limited to, tetracycline,ecdysone, steroid-responsive). The expression vector must be replicablein the host organisms either as an episome or by integration into hostchromosomal DNA. In a preferred embodiment, the expression vectorcomprises a plasmid. However, the invention is intended to include anyother suitable expression vectors, such as viral vectors.

[0422] The phrase “substantially similar” is used herein in reference tothe nucleotide sequence of DNA, or the amino acid sequence of protein,having one or more conservative or non-conservative variations from theprotease biosensor sequences disclosed herein, including but not limitedto deletions, additions, or substitutions wherein the resulting nucleicacid and/or amino acid sequence is functionally equivalent to thesequences disclosed and claimed herein. Functionally equivalentsequences will function in substantially the same manner to producesubstantially the same protease biosensor as the nucleic acid and aminoacid compositions disclosed and claimed herein. For example,functionally equivalent DNAs encode protease biosensors that are thesame as those disclosed herein or that have one or more conservativeamino acid variations, such as substitutions of non-polar residues forother non-polar residues or charged residues for similarly chargedresidues, or addition to/deletion from regions of the protease biosensornot critical for functionality. These changes include those recognizedby those of skill in the art as substitutions, deletions, and/oradditions that do not substantially alter the tertiary structure of theprotein.

[0423] As used herein, substantially similar sequences of nucleotides oramino acids share at least about 70%-75% identity, more preferably80-85% identity, and most preferably 90-95% identity. It is recognized,however, that proteins (and DNA or mRNA encoding such proteins)containing less than the above-described level of homology (due to thedegeneracy of the genetic code) or that are modified by conservativeamino acid substitutions (or substitution of degenerate codons) arecontemplated to be within the scope of the present invention.

[0424] The term “heterologous” as it relates to nucleic acid sequencessuch as coding sequences and control sequences, denotes sequences thatare not normally associated with a region of a recombinant construct,and/or are not normally associated with a particular cell. Thus, a“heterologous” region of a nucleic acid construct is an identifiablesegment of nucleic acid within or attached to another nucleic acidmolecule that is not found in association with the other molecule innature. For example, a heterologous region of a construct could includea coding sequence flanked by sequences not found in association with thecoding sequence in nature. Another example of a heterologous codingsequence is a construct where the coding sequence itself is not found innature (e.g., synthetic sequences having codons different from thenative gene). Similarly, a host cell transformed with a construct whichis not normally present in the host cell would be consideredheterologous for purposes of this invention.

[0425] Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.).

[0426] The biosensors of the present invention are constructed and usedto transfect host cells using standard techniques in the molecularbiological arts. Any number of such techniques, all of which are withinthe scope of this invention, can be used to generate proteasebiosensor-encoding DNA constructs and genetically transfected host cellsexpressing the biosensors. The non-limiting examples that followdemonstrate one such technique for constructing the biosensors of theinvention.

Example of Protease Biosensor Construction and Use

[0427] In the following examples, caspase-specific biosensors withspecific product target sequences have been constructed using sets of 4primers (2 sense and 2 antisense). These primers have overlap regions attheir termini, and are used for PCR via a primer walking technique.(Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) The two sense primers were chosen to start from the 5′polylinker (BspI) of the GFP-containing vector (Clontech, Calif.) to themiddle of the designed biosensor sequence. The two antisense primersstart from a 3′ GFP vector site (Bam HI), and overlap with the senseprimers by 12 nucleotides in the middle.

[0428] PCR conditions were as follows: 94° C. for 30 seconds fordenaturation, 55° C. for 30 seconds for annealing, and 72° C. for 30seconds for extension for 15 cycles. The primers have restrictionendonuclease sites at both ends, facilitating subsequent cloning of theresulting PCR product.

[0429] The resulting PCR product was gel purified, cleaved at BspE1 andBanH1 restriction sites present in the primers, and the resultingfragment was gel purified. Similarly, the GFP vector (Clontech, SanFrancisco, Calif.) was digested at BspE1 and BamH1 sites in thepolylinker. Ligation of the GFP vector and the PCR product was performedusing standard techniques at 16° C. overnight. E. coli cells weretransfected with the ligation mixtures using standard techniques.Transformed cells were selected on LB-agar with an appropriateantibiotic.

[0430] Cells and transfections. For DNA transfection, BHK cells andMCF-7 cells were cultured to 50-70% confluence in 6 well platescontaining 3 ml of minimal Eagle's medium (MEM) with 10% fetal calfserum, 1 mM L-glutamine, 50 μg/ml streptomycin, 50 μg/ml penicillin, 0.1mM non-essential amino acids, 1 mM sodium pyruvate and 10 μg/ml ofbovine insulin (for MCF-7 cell only) at 37° C. in a 5% CO₂ incubator forabout 36 hours. The cells were washed with serum free MEM media andincubated for 5 hours with 1 ml of transfection mixture containing 1 μgof the appropriate plasmid and 4 μg of lipofectimine (BRL) in the serumfree MEM media. Subsequently, the transfection medium was removed andreplaced with 3 ml of normal culture media. The transfected cells weremaintained in growth medium for at least 16 hours before performingselection of the stable cells based on standard molecular biologymethods (Ausubel. et al 1995).

[0431] Apoptosis assay. For apoptosis assays, the cells (BHK, MCF-7)stably transfected with the appropriate protease biosensor expressionvector were plated on tissue culture treated 96-well plates at 50-60%confluence and cultured overnight at 37° C., 5% CO₂. Varyingconcentrations of cis-platin, staurosporine, or paclitaxel in normalculture media were freshly prepared from stock and added to cell culturedishes to replace the old culture media. The cells were then observedwith the cell screening system of the present invention at the indicatedtime points either as live cell experiments or as fixed end-pointexperiments.

[0432] 1. Construction of 3-Domain Protease Biosensors

[0433] a. Caspase-3 Biosensor With an Annexin II Reactant TargetingDomain (pljkGFP).

[0434] The design of this biosensor is outlined in FIG. 31, and itssequence is shown in SEQ ID NO:1 and 2.

[0435] Primers for Caspase 3, Product Target Sequence=cone(CP3GFP-CYTO): (SEQ ID NO:153) 1) TCA TCA TCC GGA GCT GGA GCC GGA GCTGGC CGA TCG GCT GTT AAA TCT GAA GGA AAG AGA AAG TGT GAC GAA GTT GAT GGAATT GAT GAA GTA GCA (SEQ ID NO:154) 2) GAA GAA GGA TCC GGC ACT TGG GGGTGT AGA ATG AAC ACC CTC CAA GCT GAG CTT GCA CAG GAT TTC GTG GAC AGT AGACAT AGT ACT TGC TAC TTC ATC (SEQ ID NO:155) 3) TCA TCA TCC GGA GCT GGA(SEQ ID NO:156) 4) GAA GAA GGA TCC GGC ACT

[0436] This biosensor is restricted to the cytoplasm by the reactanttarget sequence. The reactant target sequence is the annexin IIcytoskeletal binding domain (MSTVHEILCKLSLEGVHSTPPSA) (SEQ ID NO:124)(FIG. 29C) (Eberhard et al. 1997. Mol. Biol. Cell 8:293a). The enzymerecognition site corresponds to two copies of the amino acid sequenceDEVD (SEQ ID NO:60) (FIG. 29B), which serves as the recognition site ofcaspase-3. Other examples with different numbers of protease recognitionsites and/or additional amino acids from a naturally occurring proteaserecognition site are shown below. The signal domain is EGFP (SEQ IDNO:46) (FIG. 29A) (Clontech, Calif.). The parent biosensor (thereactant) is restricted to the cytoplasm by binding of the annexin IIdomain to the cytoskeleton, and is therefore excluded from the nucleus.Upon cleavage of the protease recognition site by caspase 3, the signaldomain (EGFP) is released from the reactant targeting domain (annexinII), and is distributed throughout the whole volume of the cell, becauseit lacks any specific targeting sequence and is small enough to enterthe nucleus passively. (FIG. 32)

[0437] The biosensor response is measured by quantitating the effectivecytoplasm-to-nuclear translocation of the signal (see above).Measurement of the response is by one of several modes, includingintegrated or average nuclear region intensity, the ratio or differenceof the integrated or average cytoplasm intensity to integrated oraverage nuclear intensity. The nucleus is defined using a DNA-specificdye, such as Hoechst 33342.

[0438] This biosensor provides a measure of the proteolytic activityaround the annexin II cytoskeleton binding sites within the cell. Giventhe dispersed nature of the cytoskeleton and the effectively diffusestate of cytosolic enzymes, this provides an effective measure of thecytoplasm in general.

[0439] Results & Discussion:

[0440]FIG. 32 illustrates images before and after stimulation ofapoptosis by cis-platin in BHK cells, transfected with the caspase 3biosensor. The images clearly illustrate accumulation of fluorescence inthe nucleus. Generation of the spatial change in fluorescence isnon-reversible and thus the timing of the assay is flexible. Controlsfor this biosensor include using a version in which thecaspase-3-specific site has been omitted. In addition, disruption of thecytoskeleton with subsequent cell rounding did not produce the change influorescence distribution. Our experiments demonstrate the correlationof nuclear condensation with activation of caspase activity. We havealso tested this biosensor in MCF-7 cells. A recent report measured apeak response in caspase-3 activity 6 h after stimulation of MCF-7 cellswith etoposide accompanied by cleavage of PARP (Benjamin et al. 1998.Mol Pharmacol. 53:446-50). However, another recent report found thatMCF-7 cells do not possess caspase-3 activity and, in fact, thecaspase-3 gene is functionally deleted (Janicke et al. 1998. J BiolChem. 273:9357-60). Caspase-3 activity was not detected with the caspasebiosensor in MCF-7 cells after a 15 h treatment with 100 μM etoposide.

[0441] Janicke et al., (1998) also indicated that many of theconventional substrates of caspase-3 were cleaved in MCF-7 cells upontreatment with staurosporine. Our experiments demonstrate that caspaseactivity can be measured using the biosensor in MCF-7 cells when treatedwith staurosporine. The maximum magnitude of the activation bystaurosporine was approximately one-half that demonstrated withcis-platin in BHK cells. This also implies that the current biosensor,although designed to be caspase-3-specific, is indeed specific for aclass of caspases rather than uniquely specific for caspase-3. The mostlikely candidate is caspase-7 (Janicke et al., 1998). These experimentsalso demonstrated that the biosensor can be used in multiparameterexperiments, with the correlation of decreases in mitochondrial membranepotential, nuclear condensation, and caspase activation.

[0442] We have specifically tested the effects of paclitaxel on caspaseactivation using the biosensor. Caspase activity in BHK and MCF-7 cellswas stimulated by paclitaxel. It also appears that caspase activationoccurred after nuclear morphology changes. One caveat is that, based onthe above discussions, the caspase activity reported by the biosensor inthis assay is likely to be due to the combination of caspase-3 and, atleast, caspase-7 activity.

[0443] Consistent with the above results using staurosporine stimulationon MCF-7 cells, paclitaxel also stimulated the activation of caspaseactivity. The magnitude was similar to that of staurosporine. Thisexperiment used a much narrower range of paclitaxel than previousexperiments where nuclear condensation appears to dominate the response.

[0444] b. Caspase Biosensor With the Microtubule Associated Protein 4(MAP4) Projection Domain (CP8GFPNLS-SIZEPROJ)

[0445] Another approach for restricting the reactant to the cytoplasm isto make the biosensor too large to penetrate the nuclear pores Cleavageof such a biosensor liberates a product capable of diffusing into thenucleus.

[0446] The additional size required for this biosensor is provided byusing the projection domain of MAP4 (SEQ ID NO:142) (FIG. 29C)(CP8GFPNLS-SIZEPROJ). The projection domain of MAP4 does not interactwith microtubules on its own, and, when expressed, is diffuselydistributed throughout the cytoplasm, but is excluded from the nucleusdue to its size (˜120 kD). Thus, this biosensor is distinct from the oneusing the full length MAP4 sequence. (see below) One of skill in the artwill recognize that many other such domains could be substituted for theMAP4 projection domain, including but not limited to multiple copies ofany GFP or one or more copies of any other protein that lacks an activeNLS and exceeds the maximum size for diffusion into the nucleus(approximately 60 kD; Alberts, B., Bray, D., Raff, M., Roberts, K.,Watson, J. D. (Eds.) Molecular Biology of the Cell, third edition, NewYork: Garland publishing, 1994. pp 561-563). The complete sequence ofthe resulting biosensor is shown in SEQ ID NO: 3-4. A similar biosensorwith a different protease recognition domain is shown in SEQ ID NO:5-6.

[0447] c. Caspase Biosensor With a Nuclear Export Signal

[0448] Another approach for restricting the reactant to the cytoplasm isto actively restrict the reactant from the nucleus by using a nuclearexport signal. Cleavage of such a biosensor liberates a product capableof diffusing into the nucleus.

[0449] The Bacillus anthracis bacterium expresses a zinc metalloproteaseprotein complex called anthrax protease. Human mitogen activated proteinkinase kinase 1 (MEK 1) (Seger et al., J. Biol. Chem. 267:25628-25631,1992) possesses an anthrax protease recognition site (amino acids 1-13)(SEQ ID NO:102) (FIG. 29B) that is cleaved after amino acid 8, as wellas a nuclear export signal at amino acids 32-44 (SEQ ID NO:140) (FIG.29C). Human MEK 2 (Zheng and Guan, J. Biol. Chem. 268:11435-11439, 1993)possesses an anthrax protease recognition site comprising amino acidresidues 1-16 (SEQ ID NO:104) (FIG. 29B) and a nuclear export signal atamino acids 36-48. (SEQ ID NO:148) (FIG. 29C).

[0450] The anthrax protease biosensor comprises Fret25 (SEQ ID NO:48)(FIG. 29A) as the signal, the anthrax protease recognition site, and thenuclear export signal from MEK 1 or MEK2. (SEQ ID NOS: 7-8 (MEK1); 9-10(MEK2)) The intact biosensor will be retained in the cytoplasm byvirture of this nuclear export signal (eg., the reactant target site).Upon cleavage of the fusion protein by anthrax protease, the NES will beseparated from the GFP allowing the GFP to diffuse into the nucleus.

[0451] 2. Construction of 4- and 5-Domain Biosensors

[0452] For all of the examples presented above for 3-domain proteasebiosensors, a product targeting sequence, including but not limited tothose in FIG. 29C, such as a nuclear localization sequence (NLS), can beoperatively linked to the signal sequence, and thus cause the signalsequence to segregate from the reactant target domain after proteolyticcleavage. Addition of a second detectable signal domain, including butnot limited to those in FIG. 29A, operatively linked with the reactanttarget domain is also useful in allowing measurement of the reaction bymultiple means. Specific examples of such biosensors are presentedbelow.

[0453] a. 4 Domain Biosensors

[0454] 1. Caspase Biosensors With Nuclear Localization Sequences

[0455] (pcas3nlsGFP; CP3GFPNLS-CYTO):

[0456] The design of the biosensor is outlined in FIG. 33, and itssequence is shown in SEQ ID NO:11-12. PCR and cloning procedures wereperformed as described above, except that the following oligonucleotideswere used:

[0457] Primers for Caspase 3, Product Target Sequence NLS(CP3GFPNLS-CYTO): (SEQ ID NO:157) 1) TCA TCA TCC GGA AGA AGG AAA CGA CAAAAG CGA TCG GCT GTT AAA TCT GAA GGA AAG AGA AAG TGT GAC GAA GTT GAT GGAATT GAT GAA GTA GCA (SEQ ID NO:154) 2) GAA GAA GGA TCC GGC ACT TGG GGGTGT AGA ATG AAC ACC CTC CAA GCT GAG CTT GCA CAG GAT TTC GTG GAC AGT AGACAT AGT ACT TGC TAC TTC ATC (SEQ ID NO:158) 3) TCA TCA TCC GGA AGA AGG(SEQ ID NO:156) 4) GAA GAA GGA TCC GGC ACT

[0458] This biosensor is similar to that shown in SEQ ID NO:2 exceptupon recognition and cleavage of the protease recognition site, theproduct is released and the signal accumulates specifically in thenucleus due to the presence of a nuclear localization sequence, RRKRQK(SEQ ID NO:128) (FIG. 29C)(Briggs et al., J. Biol. Chem. 273:22745,1998) attached to the signal. A specific benefit of this construct isthat the products are clearly separated from the reactants. Thereactants remain in the cytoplasm, while the product of the enzymaticreaction is restricted to the nuclear compartment. The response ismeasured by quantitating the effective cytoplasm-to-nucleartranslocation of the signal, as described above.

[0459] With the presence of both product and reactant targetingsequences in the parent biosensor, the reactant target sequence shouldbe dominant prior to activation (e.g., protease cleavage) of thebiosensor. One way to accomplish this is by masking the producttargeting sequence in the parent biosensor until after proteasecleavage. In one such example, the product target sequence is functionalonly when relatively near the end of a polypeptide chain (ie: afterprotease cleavage). Alternatively, the biosensor may be designed so thatits tertiary structure masks the function of the target sequence untilafter protease cleavage. Both of these approaches include comparingtargeting sequences with different relative strengths for targeting.Using the example of the nuclear localization sequence (NLS) and annexinII sequences, different strengths of NLS have been tried with cloneselection based on cytoplasmic restriction of the parent biosensor. Uponactivation, the product targeting sequence will naturally dominate thelocalization of its associated detectable sequence domain because it isthen separated from the reactant targeting sequence.

[0460] An added benefit of using this biosensor is that the product istargeted, and thus concentrated, into a smaller region of the cell.Thus, smaller amounts of product are detectable due to the increasedconcentration of the product. This concentration effect is relativelyinsensitive to the cellular concentration of the reactant. Thesignal-to-noise ratio (SNR) of such a measurement is improved over themore dispersed distribution of biosensor #1.

[0461] Similar biosensors that incorporate either the caspase 6 (SEQ IDNO:66) (FIG. 29B) or the caspase 8 protease recognition sequence (SEQ IDNO:74) (FIG. 29B) can be made using the methods described above, butusing the following primer sets: Primers for Caspase 6, Product targetsequence = NLS (CP6GFPNLS-CYTO) (SEQ ID NO:159) 1) TCA TCA TCC GGA AGAAGG AAA CGA CAA AAG CGA TCG ACA AGA CTT GTT GAA ATT GAC AAC (SEQ IDNO:160) 2) GAA GAA GGA TCC GGC ACT TGG GGG TGT AGA ATG AAC ACC CTC CAAGCT GAG CTT GCA CAG GAT TTC GTG GAC AGT AGA CAT AGT ACT GTT GTC AAT TTC(SEQ ID NO:158) 3) TCA TCA TCC GGA AGA AGG (SEQ ID NO:156) 4) GAA GAAGGA TCC GGC ACT Primers for Caspase 8, Product target sequence = NLS(CP8GFPNLS-CYTO) (SEQ ID NO:161) 1) TCA TCA TCC GGA AGA AGG AAA CGA CAAAAG CGA TCG TAT CAA AAA GGA ATA CCA GTT GAA ACA GAC AGC GAA GAG CAA CCTTAT (SEQ ID NO:162) 2) GAA GAA GGA TCC GGC ACT TGG GGG TGT AGA ATG AACACC CTC CAA GCT GAG CTT GCA CAG GAT TTC GTG GAC AGT AGA CAT AGT ACT ATAAGG TTG CTC (SEQ ID NO:158) 3) TCA TCA TCC GGA AGA AGG (SEQ ID NO:156)4) GAA GAA GGA TCC GGC ACT

[0462] The sequence of the resulting biosensors is shown in SEQ IDNO:13-14 (Caspase 6) and SEQ ID NO: 15-16 (Caspase 8). Furthermore,multiple copies of the protease recognition sites can be inserted intothe biosensor, yielding the biosensors shown in SEQ ID NO: 17-18(Caspase 3) and SEQ ID NO:19-20 (Caspase 8).

[0463] 2. Caspase 3 Biosensor With a Second Signal Domain

[0464] An alternative embodiment employs a second signal domainoperatively linked to the reactant target domain. In this example, fulllength MAP4 serves as the reactant target sequence. Upon recognition andcleavage, one product of the reaction, containing the reactant targetsequence, remains bound to microtubules in the cytoplasm with its ownunique signal, while the other product, containing the product targetsequence, diffuses into the nucleus. This biosensor provides a means tomeasure two activities at once: caspase 3 activity using a translocationof GFP into the nucleus and microtubule cytoskeleton integrity inresponse to signaling cascades initiated during apoptosis, monitored bythe MAP4 reactant target sequence.

[0465] The basic premise for this biosensor is that the reactant istethered to the microtubule cytoskeleton by virtue of the reactanttarget sequence comprising the full length microtubule associatedprotein MAP4 (SEQ ID NO:152) (FIG. 29C) In this case, a DEVD (SEQ IDNO:60) (FIG. 29B) recognition motif is located between the EYFP signal(SEQ ID NO:44) (FIG. 29A) operatively linked to the reactant targetsequence, as well as the EBFP signal (SEQ ID NO:48) (FIG. 29A)operatively linked to the C-terminus of MAP4. The resulting biosensor isshown in SEQ ID NO:21-22.

[0466] This biosensor can also include a product targeting domain, suchas an NLS, operatively linked to the signal domain.

[0467] With this biosensor, caspase-3 cleavage still releases theN-terminal GFP, which undergoes translocation to the nucleus (directedthere by the NLS). Also, the MAP4 fragment, which is still intactfollowing proteolysis by caspase-3, continues to report on the integrityof the microtubule cytoskeleton during the process of apoptosis via thesecond GFP molecule fused to the C-terminus of the biosensor. Therefore,this single chimeric protein allows simultaneous analysis of caspase-3activity and the polymerization state of the microtubule cytoskeletonduring apoptosis induced by a variety of agents. This biosensor is alsouseful for analysis of potential drug candidates that specificallytarget the microtubule cytoskeleton, since one can determine whether aparticular drug induced apoptosis in addition to affecting microtubules.

[0468] This biosensor potentially combines a unique signal for thereactant, fluorescence resonance energy transfer (FRET) from signal 2 tosignal 1, and a unique signal localization for the product, nuclearaccumulation of signal 1. The amount of product generated will also beindicated by the magnitude of the loss in FRET, but this will be asmaller SNR than the combination of FRET detection of reactant andspatial localization of the product.

[0469] FRET can occur when the emission spectrum of a donor overlapssignificantly the absorption spectrum of an acceptor molecule. (dosRemedios, C. G., and P. D. Moens. 1995. Fluorescence resonance energytransfer spectroscopy is a reliable “ruler” for measuring structuralchanges in proteins. Dispelling the problem of the unknown orientationfactor. J Struct Biol. 115:175-85; Emmanouilidou, E., A. G.Teschemacher, A. E. Pouli, L. I. Nicholls, E. P. Seward, and G. A.Rutter. 1999. Imaging Ca(2+) concentration changes at the secretoryvesicle surface with a recombinant targeted cameleon. Curr Biol.9:915-918.) The average physical distance between the donor and acceptormolecules should be between 1 m and 10 nm with a preference of between 1nm and 6 nm. The intervening sequence length can vary considerably sincethe three dimensional structure of the peptide will determine thephysical distance between donor and acceptor. This FRET signal can bemeasured as (1) the amount of quenching of the donor in the presence ofthe acceptor, (2) the amount of acceptor emission when exciting thedonor, and/or (3) the ratio between the donor and acceptor emission.Alternatively, fluorescent lifetimes of donor and acceptor could bemeasured.

[0470] This case adds value to the above FRET biosensor by nature of theexistence of the reactant targeting sequence. This sequence allows theplacement of the biosensor into specific compartments of the cell for amore direct readout of activity in those compartments such as the innersurface of the plasma membrane.

[0471] The cytoplasmic second signal represents both original reactantplus one part of the product. The nuclear first signal representsanother product of the reaction. Thus the enzymatic reaction has theadded flexibility in that it can be represented as (1) nuclearintensity; (2) the nucleus/cytoplasm ratio; (3) the nucleus/cytoplasmFRET ratio; (4) cytoplasmic/cytoplasmic FRET ratio.

[0472] The present FRET biosensor design differs from previousFRET-based biosensors (see WO 97/28261; WO9837226) in that it signalmeasurement is based on spatial position rather than intensity. Theproducts of the reaction are segregated from the reactants. It is thischange in l spatial position that is measured. The FRET-based biosensoris based on the separation, but not to another compartment, of a donorand acceptor pair. The intensity change is due to the physicalseparation of the donor and acceptor upon proteolytic cleavage. Thedisadvantages of FRET-based biosensors are (1) the SNR is rather low anddifficult to measure, (2) the signal is not fixable. It must be recordedusing living cells. Chemical fixation, for example with formaldehyde,cannot preserve both the parent and resultant signal; (3) the range ofwavelengths are limiting and cover a larger range of the spectrum due tothe presence of two fluorophores or a fluorophore and chromophore; (4)the construction has greater limitations in that the donor and acceptormust be precisely arranged to ensure that the distance falls within 1-10nm.

[0473] Benefits of the positional biosensor includes: (1) ability toconcentrate the signal in order to achieve a higher SNR. (2) ability tobe used with either living or fixed cells; (3) only a single fluorescentsignal is needed; (4) the arrangement of the domains of the biosensor ismore flexible. The only limiting factor in the application of thepositional biosensor is the need to define the spatial position of thesignal which requires an imaging method with sufficient spatialresolution to resolve the difference between the reactant compartmentand the product compartment.

[0474] One of skill in the art will recognize that this approach can beadapted to report any desired combination of activities by simply makingthe appropriate substitutions for the protease recognition sequence andthe reactant target sequence, including but not limited to thosesequences shown in FIGS. 29A-C.

[0475] 3. Caspase 8 Biosensor With a Nucleolar Localization Domain(CP8GFPNUC-CYTO)

[0476] This approach (diagrammed in FIG. 34) utilizes a biosensor forthe detection of caspase-8 activity. In this biosensor, a nucleolarlocalization signal (RKRIRTYLKSCRRMKRSGFEMSRPIPSHLT) (SEQ ID NO:130)(FIG. 29C) (Ueki et al., Biochem. Biophys. Res. Comm. 252:97-100, 1998)was used as the product target sequence, and made by PCR using theprimers' described below. The PCR product was digested with BspEl andPvul and gel purified. The vector and the PCR product were ligated asdescribed above.

[0477] Primers for Caspase 8, Nucleolar Localization Signal(CP8GFPNUC-CYTO): (SEQ ID NO:163) 1) TCA TCA TCC GGA AGA AAA CGT ATA CGTACT TAC CTC AAG TCC TGC AGG CGG ATG AAA AGA (SEQ ID NO:164) 2) GAA GAACGA TCG AGT AAG GTG GGA AGG AAT AGG TCG AGA CAT CTC AAA ACC ACT TCT TTTCAT (SEQ ID NO:165) 3) TCA TCA TCC GGA AGA AAA (SEQ ID NO:166) 4) GAAGAA CGA TCG AGT AAG

[0478] The sequence of the resulting biosensor is shown in SEQ ID NO:23-24. This biosensor includes the protease recognition site forcaspase-8 (SEQ ID NO:74) (FIG. 29B). A similar biosensor utilizes theprotease recognition site for caspase-3. (SEQ ID NO:25-26)

[0479] These biosensors could be used with other biosensors that possessthe same product signal color that are targeted to separatecompartments, such as CP3GFPNLS-CYTO. The products of each biosensorreaction can be uniquely measured due to separation of the productsbased on the product targeting sequences. Both products fromCP8GFPNUC-CYTO and CP3GFPNLS-CYTO are separable due to the differentspatial positions, nucleus vs. nucleolus, even though the colors of theproducts are exactly the same. Assessing the non-nucleolar, nuclearregion in order to avoid the spatial overlap of the two signals wouldperform the measurement of CP3GFPNLS in the presence of CP8GFPNUC. Theloss of the nucleolar region from the nuclear signal is insignificantand does not significantly affect the SNR. The principle of assessingmultiple parameters using the same product color significantly expandsthe number of parameters that can be assessed simultaneously in livingcells. This concept can be extended to other non-overlapping producttarget compartments.

[0480] Measurement of translocation to the nucleolar compartment isperformed by (1) defining a mask corresponding to the nucleolus based ona nucleolus-specific marker, including but not limited to an antibody tonucleolin (Lischwe et al., 1981. Exp. Cell Res. 136:101-109); (2)defining a mask for the reactant target compartment, and (3) determiningthe relative distribution of the signal between these two compartments.This relative distribution could be represented by the difference in thetwo intensities or, preferably, the ratio of the intensities betweencompartments.

[0481] The combination of multiple positional biosensors can becomplicated if the reactant compartments are overlapping. Although eachsignal could be measured by simply determining the amount of signal ineach product target compartment, higher SNR will be possible if eachreactant is uniquely identified and quantitated. This higher SNR can bemaximized by adding a second signal domain of contrasting fluorescentproperty. This second signal may be produced by a signal domainoperatively linked to the product targeting sequence, or by FRET (seeabove), or by a reactant targeting sequence uniquely identifying itwithin the reactant compartment based on color, spatial position, orfluorescent property including but not limited to polarization orlifetime. Alternatively, for large compartments, such as the cytoplasm,it is possible to place different, same colored biosensors in differentparts of the same compartment.

[0482] 4. Protease Biosensors With Multiple Copies of a Second SignalDomain Serving as a Reactant Target Domain

[0483] In another example, (CP8YFPNLS-SIZECFPn) increasing the size ofthe reactant is accomplished by using multiple inserts of a secondsignal sequence, for example, ECFP (SEQ ID NO:50) (FIG. 29A) (Tsien, R.Y. 1998. Annu Rev Biochem. 67:509-44). Thus, the multiple copies of thesecond signal sequence serve as the reactant target domain by excludingthe ability of the biosensor to diffuse into the nucleus. This type ofbiosensor provides the added benefit of additional signal beingavailable per biosensor molecule. Aggegation of multiple fluorescentprobes also can result in unique signals being manifested, such as FRET,self quenching, eximer formation, etc. This could provide a uniquesignal to the reactants.

[0484] 5. Tetanus/Botulinum Biosensor With Trans-Membrane TargetingDomain

[0485] In an alternative embodiment, a trans-membrane targeting sequenceis used to tether the reactant to cytoplasmic vesicles, and analternative protease recognition site is used. The tetanus/botulinumbiosensor (SEQ ID NOS:27-28 (cellubrevin); 29-30 (synaptobrevin)consists of an NLS (SEQ ID NO:128) (FIG. 29C), Fret25 signal domain (SEQID NO:52) (FIG. 29A), a tetanus or botulinum zinc metalloproteaserecognition site from cellubrevin (SEQ ID NO:106) (FIG. 29B) (McMahon etal., Nature 364:346-349, 1993; Martin et al., J. Cell Biol., in press)or synaptobrevin (SEQ ID NO:108) (FIG. 29B) (GenBank Accession #U64520),and a trans-membrane sequence from cellubrevin (SEQ ID NO:146) (FIG.29C) or synaptobrevin (SEQ ID NO:144) (FIG. 29C) at the 3′-end whichtethers the biosensor to cellular vesicles. The N-terminus of eachprotein is oriented towards the cytoplasm. In the intact biosensor, GFPis tethered to the vesicles. Upon cleavage by the tetanus or botulinumzinc metalloprotease, GFP will no longer be associated with the vesicleand is free to diffuse throughout the cytoplasm and the nucleus.

[0486] b. 5-Domain Biosensors

[0487] 1. Caspase 3 Biosensor With a Nuclear Localization Domain and aSecond Signal Domain Operatively Linked to an Annexin II Domain

[0488] The design of this biosensor is outlined in FIG. 35, and thesequence is shown in SEQ ID NO:33-34. This biosensor differs from SEQ IDNO 11-12 by including a second detectable signal, ECFP (SEQ ID NO:50)(FIG. 29A) (signal 2) operatively linked to the reactant targetsequence.

[0489] 2. Caspase 3 Biosensor With a Nuclear Localization Sequence and aSecond Signal Domain Operatively Linked to a MAP4 Projection Domain(CP3YFPNLS-CFPCYTO)

[0490] In this biosensor (SEQ ID NO:31-32), an NLS product targetingdomain (SEQ ID NO:128) (FIG. 29C) is present upstream of an EYFP signaldomain (SEQ ID NO:44) (FIG. 29A). A DEVD protease recognition domain(SEQ ID NO:60) (FIG. 29B) is between after the EYFP signal domain andbefore the MAP4 projection domain (SEQ ID NO:142) (FIG. 29C).

EXAMPLE 11 Fluorescent Biosensor Toxin Characterization

[0491] As used herein, “toxin” refers to any organism, macromolecule, ororganic or inorganic molecule or ion that alters normal physiologicalprocesses found within a cell, or any organism, macromolecule, ororganic or inorganic molecule or ion that alters the physiologicalresponse to modulators of known physiological processes. Thus, a toxincan mimic a normal cell stimulus, or can alter a response to a normalcell stimulus.

[0492] Living cells are the targets of toxic agents that can compriseorganisms, macromolecules, or organic or inorganic molecules. Acell-based approach to toxin detection, classification, andidentification would exploit the sensitive and specific moleculardetection and amplification system developed by cells to sense minutechanges in their external milieu. By combining the evolved sensingcapability of cells with the luminescent reporter molecules and assaysdescribed herein, intracellular molecular and chemical events caused bytoxic agents can be converted into detectable spatial and temporalluminescent signals.

[0493] When a toxin interacts with a cell, whether it is at the cellsurface or within a specific intracellular compartment, the toxininvariably undermines one or more components of the molecular pathwaysactive within the cell. Because the cell is comprised of complexnetworks of interconnected molecular pathways, the effects of a toxinwill likely be transmitted throughout many cellular pathways. Therefore,our strategy is to use molecular markers within key pathways likely tobe affected by toxins, including but not limited to cell stresspathways, metabolic pathways, signaling pathways, and growth anddivision pathways.

[0494] We have developed and characterized three classes of cell basedluminescent reporter molecules to serve as reporters of toxic threatagents. These 3 classes are as follows:

[0495] (1) Detectors: general cell stress detection of a toxin;

[0496] (2) Classifiers: perturbation of key molecular pathway(s) fordetection and classification of a toxin; and

[0497] (3) Identifiers: activity mediated detection and identificationof a toxin or a group of toxins.

[0498] Thus, in another aspect of the present invention, living cellsare used as biosensors to interrogate the environment for the presenceof toxic agents. In one embodiment of this aspect, an automated methodfor cell based toxin characterization is disclosed that comprisesproviding an array of locations containing cells to be treated with atest substance, wherein the cells possess at least a first luminescentreporter molecule comprising a detector and a second luminescentreporter molecule selected from the group consisting of a classifier oran identifier; contacting the cells with the test substance eitherbefore or after possession of the first and second luminescent reportermolecules by the cells; imaging or scanning multiple cells in each ofthe locations containing multiple cells to obtain luminescent signalsfrom the detector; converting the luminescent signals from the detectorinto digital data to automatically measure changes in the localization,distribution, or activity of the detector on or in the cell, whichindicates the presence of a toxin in the test substance; selectivelyimaging or scanning the locations containing cells that were contactedwith test sample indicated to have a toxin in it to obtain luminescentsignals from the second reporter molecule; converting the luminescentsignals from the second luminescent reporter molecule into digital datato automatically measure changes in the localization, distribution, oractivity of the classifier or identifier on or in the cell, wherein achange in the localization, distribution, structure or activity of theclassifier identifies a cell pathway that is perturbed by the toxinpresent in the test substance, or wherein a change in the localization,distribution, structure or activity of the identifier identifies thespecific toxin that is present in the test substance. In a preferredembodiment, the cells possess at least a detector, a classifier, and anidentifier. In a further preferred embodiment, the digital data derivedfrom the classifier is used to determine which identifier(s) to employfor identifying the specific toxin or group of toxins.

[0499] As used herein, the phrase “the cells possess one or moreluminescent reporter molecules” means that the luminescent reportermolecule may be expressed as a luminescent reporter molecule by thecells, added to the cells as a luminescent reporter molecule, orluminescently labeled by contacting the cell with a luminescentlylabeled molecule that binds to the reporter molecule, such as a dye orantibody, that binds to the reporter molecule. The luminescent reportermolecule can be expressed or added to the cell either before or aftertreatment with the test substance.

[0500] The luminescent reporters comprising detectors, classifiers, andidentifiers may also be distributed separately into single or multiplecell types. For example, one cell type may contain a toxin detector,which, when activated by toxic activity, implies to the user that thesame toxin sample should be screened with reporters of the classifier oridentifier type in yet another population of cells identical to ordifferent from the cells containing the toxin detector.

[0501] The detector, classifier, and identifier can comprise the samereporter molecule, or they can comprise different reporters.

[0502] Screening for changes in the localization, distribution,structure or activity of the detectors, classifiers, and/or identifierscan be carried out in either a high throughput or a high content mode.In general, a high-content assay can be converted to a high-throughputassay if the spatial information rendered by the high-content assay canbe recoded in such a way as to no longer require optical spatialresolution on the cellular or subcellular levels. For example, ahigh-content assay for microtubule reorganization can be carried out byoptically resolving luminescently labeled cellular microtubules andmeasuring their morphology (e.g., bundled vs. non-bundled or normal). Ahigh-throughput version of a microtubule reorganization assay wouldinvolve only a measurement of total microtubule polymer mass aftercellular extraction with a detergent. That is, destabilizedmicrotubules, being more easily extracted, would result in a lower totalmicrotubule mass luminescence signal than unperturbed or drug-stabilizedluminescently labeled microtubules in another treated cell population.The luminescent signal emanating from a domain containing one or morecells will therefore be proportional to the total microtubule massremaining in the cells after toxin treatment and detergent extraction.

[0503] The methods for detecting, classifying, and identifying toxinscan utilize the same screening methods described throughout the instantapplication, including but not limited to detecting changes in cytoplasmto nucleus translocation, nucleus or nucleolus to cytoplasmtranslocation, receptor internalization, mitochondrial membranepotential, signal intensity, the spectral response of the reportermolecule, phosphorylation, intracellular free ion concentration, cellsize, cell shape, cytoskeleton organization, metabolic processes, cellmotility, cell substrate attachment, cell cycle events, and organellarstructure and function.

[0504] In all of these embodiments, the methods can be operated in bothtoxin-mimetic and toxin-inhibitory modes.

[0505] Such techniques to assess the presence of toxins are useful formethods including, but not limited to, monitoring the presence ofenvironmental toxins in test samples and for toxins utilized in chemicaland biological weapons; and for detecting the presence andcharacteristics of toxins during environmental remediation, drugdiscovery, clinical applications, and during the normal development andmanufacturing process by virtually any type of industry, including butnot limited to agriculture, food processing, automobile, electronic,textile, medical device, and petroleum industries.

[0506] We have developed and characterized examples of luminescentcell-based reporters, distributed across the 3 sensor classes. Themethods disclosed herein can be utilized in conjunction with computerdatabases, and data management, mining, retrieval, and display methodsto extract meaningful patterns from the enormous data set generated byeach individual reporter or a combinatorial of reporters in response totoxic agents. Such databases and bioinformatics methods include, but arenot limited to, those disclosed in U.S. patent application Ser. No.09/437,976, filed Nov. 10, 1999; No. 60/145,770 filed Jul. 27, 1999 andU.S. patent application Ser. No. ______ to be assigned, filed Feb. 19,2000. (98,068-C)

[0507] Any cell type can be used to carry out this aspect of theinvention, including prokaryotes such as bacteria and archaebacteria,and eukaryotes, such as single celled fungi (for example, yeast), molds(for example, Dictyostelium), and protozoa (for example, Euglena).Higher eukaryotes, including, but not limited to, avian, amphibian,insect, and mammalian cells can also be used.

[0508] Examples of Biosensors Response to model toxins Number Name ClassCell Types Positive Negative 1 Mito- D LLCPK (pig Valinomycin Oligomycinchondrial epithelia) (10 nM- (10 nM) Potential Rat primary 100 μM)[Donnan hepatocytes FCCP Equilibrium (10 nM- Dye] 100 μM) 2 Heat Shock DHeLa Cadmium TNF-α Protein 3T3 (10 mM) (100 ng/ml) (Hsp 27, Hsp 70)GFP-chimera 3 Tubulin- C BHK Paclitaxel Stauro- cytoskeleton HeLa (10nM- sporine [β-tubulin- LLCPK 20 μM) (1 nM- GFP Curacin-A 1 μM) chimera](5 nM- 10 μM) Nocadazole (7 nM- 12 μM) Colchicine (5 nM- 10 μM)Vinblastine (5 nM- 10 μM) 4 pp38 C 3T3 Anisomycin TNF-α MAPK- LLCPK (100μM) (100 ng/ml) stress Cadmium signaling (10 μM) [antibody and GFP-chimera] 5 NF-κB- C HeLa TNF-α Anisomycin stress 3T3 (100 ng/ml- (10 nM-signaling BHK 0.38 pg/ml) 10 μM) [antibody SNB19 IL-1 Cadmium and GFP-HepG2 (4 ng/ml- (1-10 μM) chimera] LLCPK 0.95 pg/ml) Penitrem A Nisin(10 μM) (2-1000 Valinomycin μg/ml) (1 μM) Streptolysin (10 μg/ml)Anisomycin (100 μM) 6 IκB C In many [complement cell types to NF-κB] 7Tetanus I In many Toxin cell types [Protease activity- based sensor] 8Anthrax LF I In many [Protease cell types activity- based sensor]

[0509] Examples of Detectors: This class of sensors provides a firstline signal that indicates the presence of a toxic agent. This class ofsensors provides detection of general cellular stress that requiresresolution limited only to the domain over which the measurement isbeing made, and they are amenable to high content screens as well. Thus,either high throughput or high content screening modes may be used,including but not limited to translocation of heat shock factors fromthe cytoplasm to the nucleus, and changes in mitochondrial membranepotential, intracellular free ion concentration detection (for example,Ca²⁺; H⁺), general metabolic status, cell cycle timing events, andorganellar structure and function.

[0510] 1. Mitochondrial Potential

[0511] A key to maintenance of cellular homeostasis is a constant ATPenergy charge. The cycling of ATP and its metabolites ADP, AMP,inorganic phosphate, and solution-phase protons is continuously adjustedto meet the catabolic and anabolic needs of the cell. Mitochondria areprimarily responsible for maintaining a constant energy chargethroughout the entire cell. To produce ATP from its constituents,mitochondria must maintain a constant membrane potential within theorganelle itself. Therefore, measurement of this electrical potentialwith specific luminescent probes provides a sensitive and rapid readoutof cellular stress.

[0512] We have utilized a positively charged cyanine dye, JC-1(Molecular Probes, Eugene, Oreg.), which diffuses into the cell andreadily partitions into the mitochondrial membrane, for measurement ofmitochondrial potential. The photophysics of JC-1 are such that when theprobe partitions into the mitochondrial membrane and it experiences anelectrical potential >140 mV, the probe aggregates and its spectralresponse is shifted to the red. At membrane potential values <140 mV,JC-1 is primarily monomeric and its spectral response is shifted towardthe blue. Therefore, the ratio of two emission wavelengths (645 nm and530 nm) of JC-1 partitioned into mitochondria provides a sensitive andcontinuous measure of mitochondrial membrane potential.

[0513] We have been making live cell measurements in a high throughputmode as the basis of a generalized indicator of toxic stress. The goalof our initial experiments was to determine the ratio of J-aggregates ofJC-1 dye to its monomeric form both before and after toxic stress.

[0514] Procedure

[0515] 1.Cells were plated and cultured up to overnight.

[0516] 2. Cells were stained with JC-1 (10 μg/ml) for 30 minutes at 37°C. in a CO₂ incubator.

[0517] 3. Cells were then washed quickly with HBSS at 37° C. (2 times,150 μl/well), the toxins were added if required, and the entire platewas scanned in a plate reader. The JC-1 monomer was measured optimallywith a 485 nm excitation/530 nm emission wavelength filter set, and theaggregates were best measured with a 590 nm excitation/645 nm emissionwavelength set.

[0518] Results

[0519] The mitochondrial potential within several types of living cells,and the effects of toxins on the potential were measured using thefluorescence ratio Em 645 (590)/Em 530 (485) (excitation wavelengths inparentheses). For example, we measured the effect of 10 μM valinomycinon the mitochondrial potential within LLCPK cells (pig epithelia).Within seconds of treatment, the toxin induced a more rapid and highermagnitude decrease (an approximately 50% reduction) in mitochondrialpotential than that found in untreated cells. Hepatocytes were alsodetermined to be sensitive to valinomycin, and the changes inmitochondrial potential were nearly complete within seconds to minutesafter addition of various concentrations of the toxin.

[0520] These results are consistent with mitochondrial potential being amodel intracellular detector of cell stress. Because these measurementsrequire no spatial resolution within individual cells, mitochondrialpotential measurements can be made rapidly on an entire cell array (e.g.high throughput). This means, for example, that complex arrays of manycell types can be probed simultaneously and continuously as ageneralized toxic response. Such an indicator can provide a first linesignal to indicate that a general toxic stress is present in a sample.Further assays can then be conducted to more specifically identify thetoxin using cells classifier or identifier type reporter molecules.

[0521] 2. Heat Shock Proteins

[0522] Most mammalian cells will respond to a variety of environmentalstimuli with the induction of a family of proteins called stressproteins. Anoxia, amino acid analogues, sulfhydryl-reacting reagents,transition metal ions, decouplers of oxidative phosphorylation, viralinfections, ethanol, antibiotics, ionophores, non-steroidalantiinflammatory drugs, thermal stress and metal chelators are allinducers of cell stress protein synthesis, function, or both. Uponinduction, cell stress proteins play a role in folding and unfoldingproteins, stabilizing proteins in abnormal configurations, and repairingDNA damage.

[0523] There is evidence that at least four heat shock proteinstranslocate from the cytoplasm to the nucleus upon stress activation ofthe cell. These proteins include the heat shock proteins HSP27 andHSP70, the heat shock cognate HSC70, and the heat shock transcriptionfactor HSF1. Therefore, measurement of cytoplasm to nucleartranslocation of these proteins (and other stress proteins thattranslocate from the cytoplasm to the nucleus upon a cell stress) willprovide a rapid readout of cellular stress.

[0524] We have tested the response of an HSP27-GFP biosensor (SEQ ID169-170) in two cell lines (BHK and HeLa) using a library of heavy metalchemical compounds as biological toxin stimulants to stress the cells.Briefly, cells expressing the HSP27-GFP biosensor are plated into96-well microplates, and allowed to attach. The cells are then treatedwith a panel of cell stress-inducing compounds. Exclusively cytoplasmiclocalization of the fusion protein was found in unstimulated cells.

[0525] Other similar heat shock protein biosensors (HSP-70, HSC70, andHSF1 fused to GFP) can be used as detectors, and are shown in SEQ ID NO:171-176.

[0526] Examples of Classifiers:

[0527] This class of sensors detects the presence of, and furtherclassifies toxins by identifying the cellular pathway(s) perturbed bythe toxin. As such, this suite of sensors can detect and/or classifytoxins into broad categories, including but not limited to “toxinsaffecting signal transduction,” “toxins affecting the cytoskeleton,” and“toxins affecting protein synthesis”. Either high throughput or highcontent screening modes may be used. Classifiers can comprise compoundsincluding but not limited to tubulin, microtubule-associated proteins,actin, actin-binding proteins including but not limited to vinculin,α-actinin, actin depolymerizing factor/cofilin, profilin, and myosin;NF-κB, IκB, GTP-binding proteins including but not limited to rac, rho,and cdc42, and stress-activated protein kinases including but notlimited to p38 mitogen-activated protein kinase.

[0528] 1. Tubulin-Cytoskeleton

[0529] The cell cytoskeleton plays a major role in cellular functionsand processes, such as endo- and exocytosis, vesicle transport, andmitosis. Cytoskeleton-affecting toxins, of proteinaceous andnon-proteinaceous form, such as C2 toxin, and several classes ofenterotoxins, act either directly on the cytoskeleton, or indirectly viaregulatory components controlling the organization of the cytoskeleton.Therefore, measurement of structural changes in the cytoskeleton canprovide classification of the toxin into a class ofcytoskeleton-affecting toxins. This assay can be conducted in a highcontent mode, as described previously, or in a high throughput mode. Forhigh throughput as discussed previously.

[0530] Such measurements will be valuable for identification of toxinsincluding, but not limited to anti-microtubule agents, agents thatgenerally affect cell cycle progression and cell proliferation,intracellular signal transduction, and metabolic processes.

[0531] For microtubule disruption assays, LLCPK cells stably transfectedwith a tubulin-GFP biosensor plasmid were plated on 96 well cell culturedishes at 50-60% confluence and cultured overnight at 37° C., 5% CO₂. Aseries of concentrations (10-500 nM) of 5 compounds (paclitaxel, curacinA, nocodazole, vinblastine, and colchicine) in normal culture media werefreshly prepared from stock, and were added to cell culture dishes toreplace the old culture media. The cells were then observed with thecell screening system described above, at a 12 hour time point.

[0532] Our data indicate that the tubulin chimera localizes to andassembles into microtubules throughout the cell. The microtubule arraysin cells expressing the chimera respond as follows to a variety ofanti-microtubule compounds: Drug Response Vinblastine DestabilizationNocodazole Destabilization Paclitaxel Stabilization ColchicineDestabilization Curacin A Destabilization

[0533] Similar data were obtained using cells expressing the tubulinbiosensor that were patterned onto cell arrays (such as those describedin U.S. patent application Ser. No. 08/865,341 filed May 29, 1997,incorporated by reference herein in its entirety) and dosed as above.

[0534] 2. NF-κB

[0535] NF-κB is cytoplasmic at basal levels of stimulation, but uponinsult translocates to the nucleus where it binds specific DNA responseelements and activates transcription of a number of genes. Translocationoccurs when IkB is degraded by the proteosome in response to specificphosphorylation and ubiquitination events. IkB normally retains NF-κB inthe cytoplasm via direct interaction with the protein, and masking ofthe NLS sequence of NF-κB. Therefore, although not the initial ordefining event of the whole signal cascade, NF-κB translocation to thenucleus can serve as an indicator of cell stress.

[0536] We have generated an NF-κB-GFP chimera for analysis in livecells. This was accomplished using standard polymerase chain reactiontechniques using a characterized NF-κB p65 cDNA purchased fromInvitrogen (Carlsbad, Calif.) fused to an EYFP PCR amplimer that wasobtained from Clontech Laboratories (Palo Alto, Calif.). The resultingchimera is shown in SEQ ID NO:177-178. The two PCR products were ligatedinto an eukaryotic expression vector designed to produce the chimericprotein at high levels using the ubiquitous CMV promoter.

[0537] NF-κB Immunolocalization

[0538] For further studies, we characterized endogenous NF-κB activationby immunolocalization in toxin treated cells. The NF-κB antibodies usedin this study were purchased from Santa Cruz Biotechnology, Inc. (SantaCruz, Calif.), and secondary antibodies are from Molecular Probes(Eugene, Oreg.).

[0539] For the 3T3 and SNB19 cell types, we determined the effectiveconcentrations that yield response levels of 50% of the maximum (EC50),expressed in units of mass per volume (ng/ml) and units of molarity.Based on molecular weights of 17 kD for both TNFα and IL-1α, the EC50levels for these two compounds with 3T3 and SNB19 cell types are givenin units of molarity in Table 1. Our results demonstratedreproducibility of the relative responses from zero to maximum dose, butfrom sample to sample there have been occasional shifts in the baselineintensities of the response at zero concentration.

[0540] For these experiments, either 10 or 100 TNFα-treated 3T3 or SNB19cells/well were tested. On the basis of the standard deviations measuredfor these samples, and by taking t-values for the student's t-test, wehave estimated the minimum detectable doses for each case of cell type,compound, number of cells per well, and for different choices of howmany wells are sampled per condition. The latter factor determines thenumber of degrees of freedom that are provided in the sample of data.Increasing the number of wells from 4 to 16, and increasing the numberof cells per well from 10 to 100, improves the minimum detectable dosesconsiderably. For 3T3 cells, which show lower minimum detectable dosesthan the SNB19 cells, and for the case of 1% false negative and 1% falsepositive rates, we estimate that 100 cells per well and a sampling of 12or 16 wells are sufficient to detect a dose approximately equal to theEC50 value of 0.15 ng/ml. If the false positive rate is relaxed to 20%,a concentration of approximately half that value can be detected (0.83ng/ml). One hundred cells can conveniently be sampled over a cellculture surface area of less than 1 mm². TABLE 1 EC50 levels for TNFαand IL-1α (based on molecular weights of 17 kD for both) Compound CellType EC50 (10⁻¹² moles/liter) TNFα 3T3 8.8 SNB19 5.9 IL-1α 3T3  0.24SNB19 59  

[0541] 3. Phospho-p38 Mitogen Activated Protein Kinase (pp38MAPK)

[0542] MAPKs play a role in not only cell growth and division, but asmediators of cellular stress responses. One MAPK, p38, is activated bychemical stress inducers such as hyperosmolar sorbitol, hydrogenperoxide, arsenite, cadmium ions, anisomycin, sodium salicylate, andLPS. Activation of p38 is also accompanied by its translocation into thenucleus from the cytoplasm.

[0543] MAPK p38 lies in a pathway that is a cascade of kinases. Thus,p38 is a substrate of one or more kinases, and it acts to phosphorylateone or more substrates in time and space within the living cell.

[0544] The assay we present here measures, as one of its parameters, p38activation using immunolocalization of the phosphorylated form of p38 intoxin-treated cells. The assay was developed to be flexible enough toinclude the simultaneous measurement of other parameters within the sameindividual cells. Because the signal transduction pathway mediated bythe transcription factor NF-κB is also known to be involved in the cellstress response, we included the activation of NF-κB as a secondparameter in the same assay.

[0545] Our experiments demonstrate an immunofluorescence approach can beused to measure p38 MAPK activation either alone or in combination withNF-κB activation in the same cells. Multiple cell types, model toxins,and antibodies were tested, and significant stimulation of both pathwayswas measured in a high-content mode. The phospho-p38 antibodies used inthis study were purchased from Sigma Chemical Company (St. Louis, Mo.).We report that at least two cell stress signaling pathways can not onlybe measured simultaneously, but are differentially responsive to classesof model toxins. FIG. 36 shows the differential response of the p38 MAPKand NF-κB pathways across three model toxins and two different celltypes. Note that when added alone, three of the model toxins (IL1α, TNFαand Anisomycin) can be differentiated by the two assays as activators ofspecific pathways.

[0546] IκB Chimera

[0547] IkB degradation is the key event leading to nuclear translocationof NF-kB and activation of the NFkB-mediated stress response. We havechosen this sensor to complement the NF-kB sensor as a classifier in ahigh-throughput mode: the measurement of loss of signal due todegradation of the IκB-GFP fusion protein requires no spatial resolutionwithin individual cells, and as such we envision IkB degradationmeasurements being made rapidly on an entire cell substrate.

[0548] This biosensor is based on fusion of the first 60 amino acids ofIkB to the Fred25 variant of GFP. SEQ ID 179-180 This region of IkBcontains all the regulatory sequences, including phosphorylation sitesand ubiquitination sites, necessary to confer proteosome degradationupon the biosensor. Knowing this, stimulation of any pathway that wouldtypically lead to NFkB translocation results in degradation of thisbiosensor. Monitoring the fluorescence intensity of cells expressingIkB-GFP identifies the degradation process.

[0549] Examples of Identifiers:

[0550] In our toxin identification strategy, the first two levels ofcharacterization ensure a rapid readout of toxin class withoutsacrificing the ability to detect many new mutant toxins or dissectseveral complex mixtures of known toxins. The third level of biosensorsare identifiers, which can identify a specific toxin or group of toxins.In one embodiment, an identifier comprises a protease biosensor thatresponds to the activity of a specific toxin. Other identifiers areproduced with reporters/biosensors specific to their activities. Theseinclude, but are not limited to post-translational modifications such asphosphorylation or ADP-ribosylation, translocation between cellularorganelles or compartments, effects on specific organelles or cellularcomponents (for example, membrane permeabilization, cytoskeletonrearrangement, etc.)

[0551] ADP-ribosylatin toxins—These toxins include Pseudomonas toxin A,diptheria toxin, botulinum toxin, pertussis toxin, and cholera toxin.For example, C. botulinum C2 toxin induces the ADP-ribosylation ofArg177 in the cytoskeletal protein actin, thus altering its assemblyproperties. Besides the construction of a classifier assay to measureactin-cytoskeleton regulation, an identifier assay can be constructed todetect the specific actin ADP-ribosylation. Because the ADP-ribosylationinduces a conformational change that no longer permits the modifiedactin to polymerize, this conformational change can be detectedintracellularly in several possible ways using luminescent reagents. Forexample, actin can be luminescently labeled using a fluorescent reagentwith an appropriate excited state lifetime that allows for themeasurement of the rotational diffusion of the intracellular actin usingsteady state fluorescence anisotropy. That is, toxin-modified actin willno longer be able to assemble into rigid filaments and will thereforeproduce only luminescent signals with relatively low anisotropy, whichcan be readily measured with an imaging system. In another embodiment,actin can be labeled with a polarity-sensitive fluorescent reagent thatreports changes in actin-conformation through spectral shifts of theattached reagent. That is, toxin-treatment will induce a conformationalchange in intracellular actin such that a ratio of two fluorescencewavelengths will provide a measure of actin ADP-ribosylation.

[0552] Cytotoxic phospholipases—Several gram-positive bacterial speciesproduce cytotoxic phospholipases. For example, Clostridium perfringensproduces a phospholipase C specific for the cleavage ofphosphoinositides. These phosphoinositides (e.g., inositol1,4,5-trisphosphate) induce the release of calcium ions fromintracellular organelles. An assay that can be conducted as eitherhigh-content or high-throughput can be constructed to measure therelease of calcium ions using fluorescent reagents that have alteredspectral properties when complexed with the metal ion. Therefore, adirect consequence of the action of a phospholipase C based toxin can bemeasured as a change in cellular calcium ion concentration.

[0553] Exfoliative toxins—These toxins are produced by severalStaphylococcal species and can consist of several serotypes. A specificidentifier for these toxins can be constructed by measuring themorphological changes in their target organelle, the desmosome, whichoccur at the junctions between cells. The exfoliative toxins are knownto change the morphology of the desmosomes into two smaller componentscalled hemidesmosomes. In the high-content assay for exfoliative toxins,epithelial cells whose desmosomes are luminescently labeled aresubjected to image analysis. An method that detects the morphologicalchange between desmosomes and hemidesmosomes is used to quantify theactivity of the toxins on the cells.

[0554] Most of these identifiers can be used in high throughput assaysrequiring no spatial resolution, as well as in high content assays.

[0555] Several biological threat agents act as specific proteases, andthus we have focused on the development of fluorescent proteinbiosensors that report the proteolytic cleavage of specific amino acidsequences found within the target proteins.

[0556] A number of such protease biosensors (including FRET biosensors)are disclosed above, such as the caspase biosensors, anthrax, tetanus,Botulinum, and the zinc metalloproteases. FRET is a powerful techniquein that small changes in protein conformation, many of which areassociated with toxin activity, can not only be measured with highprecision in time and space within living cells, but can be measured ina high-throughput mode, as discussed above.

[0557] As described above, one of skill in the art will recognize thatthe protease biosensors of this aspect of the invention can be adaptedto report the activity of any protease, by a substitution of theappropriate protease recognition site in any of the constructs (see FIG.29B). As disclosed above, these biosensors can be used in high-contentor high throughput screens to detect in vivo activation of enzymaticactivity by toxins, and to identify specific activity based on cleavageof a known recognition motif. These biosensors can be used in both livecell and fixed end-point assays, and can be combined with additionalmeasurements to provide a multi-parameter assay.

[0558] Anthrax LF

[0559] Anthrax is a well-known agent of biological warfare and is anexcellent target for development of a biosensor in the identifier class.Lethal factor (LF) is one of the protein components that confer toxicityto anthrax, and recently two of its targets within cells wereidentified. LF is a metalloprotease that specifically cleaves Mek1 andMek2 proteins, kinases that are part of the MAP-kinase signalingpathway. Construction of lethal factor protease biosensors are describedabove. (SEQ ID NO:7-8; 9-10) Green fluorescent protein (GFP) is fusedin-frame at the amino terminus of either Mek1 or Mek2 (or both),resulting in a chimeric protein that is retained in the cytoplasm due tothe presence of a nuclear export sequence (NES) present in both of thetarget molecules. Upon cleavage by active lethal factor, GFP is releasedfrom the chimera and is free to diffuse into the nucleus. Therefore,measuring the accumulation of GFP in the nucleus provides a directmeasure of LF activity on its natural target, the living cell.

[0560] While a preferred form of the invention has been shown in thedrawings and described, since variations in the preferred form will beapparent to those skilled in the art, the invention should not beconstrued as limited to the specific form shown and described, butinstead is as set forth in the claims.

We claim:
 1. An automated method for cell based toxin characterizationcomprising providing an array of locations containing cells to betreated with a test substance, wherein the cells possess at least afirst luminescent reporter molecule comprising a detector and a secondluminescent reporter molecule selected from the group consisting of aclassifier or an identifier; contacting the cells with the testsubstance either before or after possession of the first and secondluminescent reporter molecules by the cells; wherein the localization,distribution, structure, or activity of the first and second luminescentreporter molecule is modified when the cell is contacted with the toxin,imaging or scanning multiple cells in each of the locations containingmultiple cells to obtain luminescent signals from the detector;converting the luminescent signals from the detector into digital data;utilizing the digital data from the detector to automatically measurethe localization, distribution, structure or activity of the detector onor in the cell, wherein a change in the localization, distribution,structure or activity of the detector indicates the presence of a toxinin the test substance; selectively imaging or scanning the locationscontaining cells that were contacted with test sample indicated to havea toxin in it to obtain luminescent signals from the second reportermolecule; converting the luminescent signals from the second luminescentreporter molecule into digital data; utilizing the digital data from thesecond luminescent reporter molecule to automatically measure thelocalization, distribution, structure, or activity of the classifier oridentifier on or in the cell, wherein a change in the localization,distribution, structure or activity of the classifier identifies a cellpathway that is perturbed by the toxin present in the test substance, orwherein a change in the localization, distribution, structure oractivity of the identifier identifies the specific toxin or group oftoxins that are present in the test substance.
 2. The method of claim 1wherein the second luminescent reporter molecule is a classifier, andthe digital data derived from the classifier is used to select anappropriate identifier for identification of the specific toxin or groupof toxins.
 3. An automated method for cell based toxin characterizationcomprising providing an array of locations containing cells to betreated with a test substance, wherein the cells possess at least afirst luminescent reporter molecule comprising a detector, a secondluminescent reporter molecule comprising a classifier, and a thirdluminescent reporter molecule comprising an identifier; contacting thecells with the test substance either before or after possession of thefirst second, and third luminescent reporter molecules by the cells;wherein the localization, distribution, structure, or activity of thefirst, second, and third luminescent reporter molecules is modified whenthe cell is contacted with the toxin, imaging or scanning multiple cellsin each of the locations containing multiple cells to obtain luminescentsignals from the detector; converting the luminescent signals from thedetector into digital data; utilizing the digital data from the detectorto automatically measure the localization, distribution, structure oractivity of the detector on or in the cell, wherein a change in thelocalization, distribution, structure or activity of the detectorindicates the presence of a toxin in the test substance; selectivelyimaging or scanning the locations containing cells that were contactedwith test sample indicated to have a toxin in it to obtain luminescentsignals from the classifier; converting the luminescent signals from theclassifier into digital data; utilizing the digital data from theclassifier to automatically measure the localization, distribution,structure, or activity of the classifier on or in the cell, wherein achange in the localization, distribution, structure or activity of theclassifier identifies a cell pathway that is perturbed by the toxinpresent in the test substance; selectively imaging or scanning thelocations containing cells that were contacted with test sampleindicated to have a toxin in it to obtain luminescent signals from theidentifier; converting the luminescent signals from the identifier intodigital data; and utilizing the digital data from the identifier toautomatically measure the localization, distribution, structure, oractivity of the identifier on or in the cell, wherein a change in thelocalization, distribution, structure or activity of the identifieridentifies the specific toxin or group of toxins that is present in thetest substance.
 4. The method of claim 3 wherein the digital dataderived from the classifier is used to select an appropriate identifierfor identification of the specific toxin or group of toxins.
 5. Themethod of any one of claim 1-4 wherein the detector comprises a moleculeselected from the group consisting of heat shock proteins and compoundsthat respond to changes in mitochondrial membrane potential,intracellular free ion concentration, cytoskeletal organization, generalmetabolic status, cell cycle timing events, and organellar structure andfunction.
 6. The method of any one of claim 1-5 wherein the classifiercomprises a molecule selected from the group consisting of tubulin,microtubule-associated proteins, actin, actin-binding proteins, NF-κB,IκB, and stress-activated kinases.
 7. The method of any one of claim 1-6wherein the cell pathway is selected from the group consisting of cellstress pathways, cell metabolic pathways, cell signaling pathways, cellgrowth pathways, and cell division pathways.
 8. The method of claim 1,wherein the second luminescent reporter molecule is an identifier, andthe identifier identifies a toxin or group of toxins selected from thegroup consisting of proteases, ADP-ribosylating toxins, cytotoxicphospholipases, and exfoliative toxins.
 9. The method of any one ofclaim 3-7, wherein the identifier identifies a toxin or group of toxinsselected from the group consisting of proteases, ADP-ribosylatingtoxins, cytotoxic phospholipases, and exfoliative toxins.
 10. The methodof any of claims 1-9 wherein the change in the localization,distribution, structure or activity of the first, second, or thirdluminescent reporter molecules is selected from the group consisting ofcytoplasm to nucleus translocation, nucleus or nucleolus to cytoplasmtranslocation, receptor internalization, mitochondrial membranepotential, loss of signal, the spectral response of the reportermolecule, phosphorylation, intracellular free ion concentration, cellsize, cell shape, cytoskeleton organization, metabolic processes, cellmotility, cell substrate attachment, cell cycle events, and organellarstructure and function.
 11. The method of any one of claims 1-10,wherein the imaging or scanning multiple cells in each of the locationscontaining multiple cells to obtain luminescent signals from thedetector is carried out in a high throughput mode.
 12. The method of anyone of claims 1-10, wherein the imaging or scanning multiple cells ineach of the locations containing multiple cells to obtain luminescentsignals from the detector is carried out in a high content mode.
 13. Themethod of claim 1-10 wherein the selective imaging or scanning of thelocations containing cells that were contacted with test sampleindicated to have a toxin in it to obtain luminescent signals from thesecond or third reporter molecule is carried out in a high throughputmode.
 14. The method of claim 1-10 wherein the selective imaging orscanning of the locations containing cells that were contacted with testsample indicated to have a toxin in it to obtain luminescent signalsfrom the second or third reporter molecule is carried out in a highcontent mode.
 15. The method of any one of claims 1-14 furthercomprising providing a digital storage media for data storage andarchiving.
 16. The method of claim 15 further comprising a means forautomated control, acquisition, processing and display of results.
 17. Acomputer readable storage medium comprising a program containing a setof instructions for causing a cell screening system to execute themethod of any one of claims 1-16, wherein the cell screening systemcomprises an optical system with a stage adapted for holding a platecontaining cells, a means for moving the stage or the optical system, adigital camera, a means for directing light emitted from the cells tothe digital camera, and a computer means for receiving and processingthe digital data from the digital camera.
 18. A kit for cell based toxindetection comprising: (a) at least one reporter molecule, wherein thelocalization, distribution, structure, or activity of the reportermolecule is modified when the cell is contacted with a toxin; (b)instructions for using the reporter molecule to carry out the method ofany one of claims 1-16 to detect toxins in a test substance.
 19. The kitof claim 18 further comprising the computer readable storage medium ofclaim
 17. 20. An automated method for cell based toxin characterizationcomprising providing a first array of locations containing cells to betreated with a test substance, wherein the cells possess a least a firstluminescent reporter molecule comprising a reporter molecule selectedfrom the group consisting of detectors and classifiers; contacting thecells with the test substance either before or after possession of thefirst luminescent reporter molecule by the cells; wherein thelocalization, distribution, structure, or activity of the firstluminescent reporter molecule is modified when the cell is contactedwith the toxin, imaging or scanning multiple cells in each of thelocations containing multiple cells to obtain luminescent signals fromthe detector; converting the luminescent signals from the detector intodigital data; utilizing the digital data from the detector toautomatically measure the localization, distribution, structure oractivity of the detector on or in the cell, wherein a change in thelocalization, distribution, structure or activity of the detectorindicates the presence of a toxin in the test substance, providing asecond array of locations containing cells to be treated with the testsubstance, wherein the cells possess a least a second luminescentreporter molecule comprising a reporter molecule selected from the groupconsisting of classifiers and identifiers, and wherein the second arrayof locations containing cells can comprise either the same or adifferent cell type as the first array of locations containing cells;contacting the second array of locations containing cells with the testsubstance either before or after possession of the second luminescentreporter molecule by the cells; wherein the localization, distribution,structure, or activity of the second luminescent reporter molecule ismodified when the cell is contacted with the toxin; utilizing thedigital data from the second luminescent reporter molecule toautomatically measure the localization, distribution, structure, oractivity of the classifier or identifier on or in the cell, wherein achange in the localization, distribution, structure or activity of theclassifier identifies a cell pathway that is perturbed by the toxinpresent in the test substance, or wherein a change in the localization,distribution, structure or activity of the identifier identifies thespecific toxin or group of toxins that are present in the testsubstance.